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Vol.:(0123456789) European Journal of Applied Physiology (2025) 125:277–301 https://doi.org/10.1007/s00421-024-05642-9 INVITED REVIEW Effects of thermal interventions on skeletal muscle adaptations and regeneration: perspectives on epigenetics: a narrative review Tom Normand‑Gravier2,5 · Robert Solsona5 · Valentin Dablainville2,3 · Sébastien Racinais4 · Fabio Borrani1 · Henri Bernardi2 · Anthony M. J. Sanchez1,5 Received: 16 July 2024 / Accepted: 12 October 2024 / Published online: 28 November 2024 © The Author(s) 2024 Abstract Recovery methods, such as thermal interventions, have been developed to promote optimal recovery and maximize long- term training adaptations. However, the beneficial effects of these recovery strategies remain a source of controversy. This narrative review aims to provide a detailed understanding of how cold and heat interventions impact long-term training adaptations. Emphasis is placed on skeletal muscle adaptations, particularly the involvement of signaling pathways regulating protein turnover, ribosome and mitochondrial biogenesis, as well as the critical role of satellite cells in promoting myofiber regeneration following atrophy. The current literature suggests that cold interventions can blunt molecular adaptations (e.g., protein synthesis and satellite cell activation) and oxi-inflammatory responses after resistance exercise, resulting in diminished exercise-induced hypertrophy and lower gains in isometric strength during training protocols. Conversely, heat interventions appear promising for mitigating skeletal muscle degradation during immobilization and atrophy. Indeed, heat treatments (e.g., passive interventions such as sauna-bathing or diathermy) can enhance protein turnover and improve the maintenance of muscle mass in atrophic conditions, although their effects on uninjured skeletal muscles in both humans and rodents remain controversial. Nonetheless, heat treatment may serve as an important tool for attenuating atrophy and pre- serving mitochondrial function in immobilized or injured athletes. Finally, the potential interplay between exercise, thermal interventions and epigenetics is discussed. Future studies must be encouraged to clarify how repeated thermal interventions (heat and cold) affect long-term exercise training adaptations and to determine the optimal modalities (i.e., method of appli- cation, temperature, duration, relative humidity, and timing). Keywords Heat treatment · Cryotherapy · Skeletal muscle · Exercise · Resistance and endurance training · Protein synthesis Abbreviations CSA Cross-sectional area CWI Cold-water immersion Communicated by Michael I Lindinger. Tom Normand-Gravier and Robert Solsona contributed equally to this work as first authors. * Anthony M. J. Sanchez anthony.sanchez@unil.ch Tom Normand‑Gravier tom.normand-gravier@univ-perp.fr Robert Solsona robert.solsona@univ-perp.fr Valentin Dablainville valentin.dablainville@aspetar.com Sébastien Racinais sebastien.racinais@creps-montpellier.sports.gouv.fr Fabio Borrani fabio.borrani@unil.ch Henri Bernardi henri.bernardi@inrae.fr 1 Institute of Sport Sciences, University of Lausanne, Lausanne, Switzerland 2 UMR866, Dynamique du Muscle et Métabolisme (DMeM), INRAE, University of Montpellier, Montpellier, France 3 Research and Scientific Support Department, Aspetar Orthopedic and Sports Medicine Hospital, 29222 Doha, Qatar 4 Environmental Stress Unit, CREPS Montpellier— Font-Romeu, Montpellier, France 5 Laboratoire Interdisciplinaire Performance Santé Environnement de Montagne (LIPSEM), Faculty of Sports Sciences, University of Perpignan Via Domitia, UR 4640, 7 Avenue Pierre de Coubertin, 66120 Font‑Romeu, France

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278 European Journal of Applied Physiology (2025) 125:277–301 CK Creatine kinase DNMT DNA methyltransferase DOMS Delayed-onset muscle soreness EIMD Exercise-induced muscle damage FOXOs Forkhead box class O proteins H2O2 Hydrogen peroxide HSP Heat shock protein HWI Hot-water immersion IL-6 Interleukin-6 miRNAs MicroRNAs MAFbx Muscle atrophy F-box gene MTORC1 Mammalian/mechanistic target of rapamy- cin complex 1 NO Nitric oxide NOS Nitrogen oxide species PDK Pyruvate dehydrogenase kinase PGC-1alpha Peroxisome proliferator-activated receptor- gamma coactivator-1alpha PPAR-delta Peroxisome proliferator-activated receptor-delta ROS Reactive oxygen species SREBF2 Sterol regulatory element binding transcrip- tion factor 2 TET Ten–eleven translocation TNF-alpha Tumor necrosis factor-alpha ̇VO2max Maximal oxygen consumption WBC Whole-body cryotherapy Introduction Physical exercise generates high metabolic and mechani- cal stress in an intensity- and duration-dependent manner, leading to structural damage to muscle cells and heightened inflammation and soreness (Malm 2001). According to the athlete background, prolonged and/or intense exercise can induce metabolite accumulation, a decrease in glyco- gen content and hyperthermia, temporarily disrupting the homeostasis of the organism (Morris et al. 2005; Fiorenza et al. 2019). A single bout of exercise can cause microscopic tears and delayed-onset muscle soreness (DOMS) (Cheung et al. 2003), with mechanical strain identified as a critical contributor to muscle damage (Newham et al. 1983; Abood- arda et al. 2011). Insufficient recovery time between train- ing sessions can lead to overtraining, accumulated fatigue and increased risk of injury (Walters et al. 2018; Méline et al. 2019; Symons et al. 2023). Exercise-induced muscle damage and/or excessive fatigue can be detrimental to per- formance and prolong recovery time. Therefore, various recovery strategies have been explored in recent decades, including nutritional interventions and physiological treat- ments like massage, active recovery, and thermal interven- tions (Kellmann et al. 2018). Among physiological recovery methods, thermal exposures have gained increased attention. Cryotherapy (from the Greek ‘cryo’ meaning cold) has been the most investigated in recent years and is one of the most popular methods used by athletes (Frery et al. 2023). Cryo- therapy refers to different cooling strategies such as the use of ice packs (≈ 0 to 2 °C), cooling garments, phase-change materials, whole-body cryotherapy (WBC, generally ≈ −110 to −130 °C) involving extreme cold air exposure, or cold- water immersion (CWI, generally ≈ 5 to 20 °C) (Chaillou et al. 2022). Conversely, methods that can be called ‘kau- matherapy’ (from the Greek ‘kauma’ meaning heat) (Méline et al. 2017), are gaining interest. Sauna bathing (80–100 °C), hot-water immersion (HWI, 38–44 °C), microwave dia- thermy (muscle temperature elevated at 38–39 °C), heat/ steam sheets (muscle temperature elevated at 38–39 °C), are heat interventions commonly used by athletes during rest periods (McGorm et al. 2018). Recent reviews have questioned the efficacy of short-term thermal interventions on recovery from exercises (Chaillou et al. 2022; Moore et al. 2022; Choo et al. 2022). Chaillou and colleagues highlighted that cold interventions might be beneficial in the early recovery phase (< 1 h post-exercise) to enhance the recovery of maximal strength (Chaillou et al. 2022) (Fig. 1a), even if the vasoconstrictor effect of cold rep- resents a confounding factor since it could limit the cellular export of biomarkers of muscle damages. Furthermore, there are still conflicting results, as most of the studies suggest that post-exercise cryotherapy does not acutely impact the recovery of maximal strength (Pointon et al. 2011; Roberts et al. 2015a; Argus et al. 2017). The effects of cryotherapy on muscle soreness seem to stem from its analgesic proper- ties (Meeusen and Lievens 1986). In this sense, a meta-anal- ysis showed that CWI (5–13 °C, 10–24 min) significantly reduces DOMS 24, 48 and 96 h post-exercise (Hohenauer et al. 2015) (Fig. 1a, c). Nevertheless, a discrepancy between this subjective recovery indicator (i.e., perceived soreness) and the objective parameters (i.e., blood plasma markers, such as interleukin-6 (IL-6) or Lactate dehydrogenase) has been noted (Vaile et al. 2008). Thus, the effects of cryo- therapy on objective recovery markers (e.g., blood lactate, creatine kinase and cytokine levels) remain controversial and present methodological limitations (i.e., unblinded, selection bias, random sequence generation etc.) (Hohenauer et al. 2015). However, since the benefits of cryotherapy include subjective parameters, it is plausible to consider that a pla- cebo effect may contribute to these effects, as suggested by Broatch and colleagues (Broatch et al. 2014). Furthermore, there is growing evidence that passive muscle heating may elicit several cellular adaptations. Combined with resistance exercise, passive muscle heating can increase markers of protein synthesis (Fig. 1f), such as the phosphorylation level of kinases involved in the MTORC1 (mammalian/mechanis- tic target of rapamycin complex 1) axis (Kakigi et al. 2011),

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279 European Journal of Applied Physiology (2025) 125:277–301 a critical pathway regulating mRNA translation (Sanchez et al. 2019; Solsona and Sanchez 2020). Passive muscle heating increases the phosphorylation level of Akt, MTOR, 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) and ribosomal protein S6 (RPS6) in human skel- etal muscle (Kakigi et al. 2011). These markers play a major role in growth, proliferation, and cellular survival (Sanchez et al. 2012a). However, the effects of acute HWI on other physiological variables appear inconsistent, with studies reporting contradictory findings on heart rate, muscle sore- ness, sprint performance, and time trial race (Kuligowski et al. 1998; Versey et al. 2013). Thus, drawing definitive conclusions about the effectiveness of acute heat interven- tion remains challenging, despite many studies suggesting it is beneficial. The effectiveness of thermal interventions in enhancing endurance and repeated sprint performance has been studied in twice-daily training sessions involving active individuals and elite athletes, respectively. In one study, heat accelerated recovery, whereas cold had an adverse effect after an exhaustive endurance exercise, as objectified by the mean power outputs during a second exercise session on the same day (Cheng et al. 2017). Moreover, in single muscle mice fibers, glycogen resynthesis is accelerated with heating (36 °C) (Cheng et al. 2017). The authors conclude that heating allows a faster recovery due to an increase of glycogen resynthesis rate (Fig. 1b). Consistent with these findings, repeated sprint performance was higher for active recovery and HWI compared to CWI, after high-intensity exercise training in elite short-track speed skaters (Solsona et al. 2023) (Fig. 1b). Importantly, maximal power was cor- related with average muscle temperature during HWI, CWI and active recovery, and mean power was correlated with muscle temperature for active recovery and CWI (Solsona et al. 2023). This suggests a direct effect of muscle tempera- ture modulation on exercise performance. Furthermore, it was recently found that heat and cold combination (from 3 to 45 °C) with compression (from 25 to 75 mmHg) for 10 or 20 min, improves muscle function (i.e., biomechanical changes, pain threshold, muscle strength, and tissue perfu- sion) in mixed martial arts athletes (Trybulski et al. 2024). These findings strongly suggests that contrast temperature interventions might be a promising tool for optimizing recovery in athletes. Several factors may contribute to the variability in the effects of thermal interventions, including Effects of cold and heat interventions on training adaptations and muscle function Post-exercise short-term recovery < 24 h Post-exercise mid-term recovery 24 h to 48 h Repeated thermal interventions > 1 week DOMS Glycogen resynthesis Post-EIMD RFD DOMS Concentric peak torque Hypertrophy Available energy RSA + HI endurance performance MVIC Isometric strength Ribosome biogenesis Satellite cell dynamics and MTORC1 pathway Protein synthesis a. Volemia Endurance performance b. c. d. e. f. g. Fig. 1 Effects of thermal interventions on training adaptations and muscle function. After a single exercise, post-exercise cold stress can decrease DOMS (delayed-onset muscle soreness) after EIMD (exercise-induced muscle damage), promoting the recovery of MVIC (maximal voluntary isometric contraction) (a). After a single bout of exercise, post-exercise heat intervention promotes glycogen resyn- thesis, accelerating the recovery of repeated sprint ability (RSA) and high-intensity (HI) endurance performance (b). During mid-term recovery (24–48 h), cold intervention can decrease DOMS (c), while heat intervention can reduce the EIMD-induced decline in the rate of force development (RFD) and concentric peak torque (d). Repeated exposures to cold intervention blunt chronic skeletal muscle adapta- tions by inhibiting the MTORC1 pathway, impairing ribosome bio- genesis, and altering satellite cell dynamics, leading to a diminished protein synthesis, hypertrophy and isometric strength (e). In rodents, repeated exposures to heat intervention promote chronic skeletal mus- cle adaptations by activating the MTORC1 pathway, ribosome bio- genesis, increase of satellite cell dynamics and protein synthesis (f). In athletes, repeated exposures to heat intervention can increase max- imal isometric strength and endurance performance (g). However, lit- erature does not allow to provide definitive conclusions regarding the impact of heat intervention on skeletal muscle mass and properties. Further studies are needed in humans to determine the optimal heat dose (e.g., temperature and duration) to maximize long-term training adaptations

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280 European Journal of Applied Physiology (2025) 125:277–301 the characteristics of the population studied (i.e., athletes, sedentary subjects, seniors, immobilized patients), the exer- cise (i.e., endurance, sprint, resistance exercise) and the therapeutic dose (i.e., timing, duration of application, treat- ment modality), and inter-individual variability such as vari- ations in fat mass percentage. Due to their potentially tran- sient nature, understanding the effects of a single thermal intervention is crucial for gaining insights into long-term adaptations. Despite the evidence concerning short interven- tion, the effects of the regular use of thermal interventions on long-term adaptations to exercise training remain poorly studied. It remains essential to ensure that these recovery strategies do not interfere with cellular adaptations involved in gains of physical aptitudes. Training interventions induce both quantitative and quali- tative adaptations in skeletal muscle, such as an increase in fiber size (resistance training) and enhanced cellular oxi- dative capacity (endurance training) (Sanchez et al. 2019). Importantly, exercise-induced cellular stress promotes physi- ological adaptations, leading to performance improvements (Kenttä and Hassmén 1998). Exercise training elicits a wide range of muscle adaptations, including modifications in the structural, metabolic and functional characteristics of myofibers. The phenotype of skeletal muscle is highly plastic and adapts to different modes of exercise. Eccen- tric exercise comprises lengthening actions and provokes sarcomere strain, notably in type II fibers (Qaisar et al. 2016). Resistance exercise particularly promotes hypertro- phy through an increase in fiber cross-sectional area (CSA) and strength gains (Lopez et al. 2021). The magnitude of the increase in muscle mass can be influenced by training volume (Schoenfeld et al. 2017), although it is important to note that relying solely on training volume as a meas- ure of training load presents limitations (Normand-Gravier et al. 2022a). For instance, high-volume resistance training leads to sarcoplasmic hypertrophy and limited strength gains (Reggiani and Schiaffino 2020). MTORC1 plays a major role in mediating hypertrophic response to resistance exercise by upregulating muscle protein synthesis rates, ribosome biogenesis, and satellite cell recruitment (Wackerhage et al. 2019; Jin et al. 2019) (Fig. 1e, f). Endurance exercise elic- its different adaptations in skeletal muscle, such as a shift from type II (fast twitch fiber) to type I (slow twitch fiber) muscle fibers (Wilson et al. 2012), mitochondrial adapta- tions and an increase in skeletal muscle capillary density (MacInnis and Gibala 2017). Importantly, recent research has shed light on the involvement of epigenetic factors in these adaptations. Epigenetics refers to mechanisms that regulate gene expression without altering the nucleotide sequence of genes (Ehlert et al. 2013; Widmann et al. 2019). Exercise, nutrition and temperature are among the behavio- ral and environmental stressors that can induce epigenetic modifications (Widmann et al. 2019; Bartke and Schneider 2020). These mechanisms are critical for cellular adaptation to environmental stress (Solsona and Sanchez 2021; Solsona et al. 2022). Thus, DNA methylation, histone modifications, and expression of non-coding RNAs such as microRNAs (miRNAs, i.e., small single-stranded molecules of about 22 nucleotides) are epigenetic mechanisms that may contribute to physiological adaptations induced by exercise or ther- mal exposure (Sajjanar et al. 2019; Widmann et al. 2019; Normand-Gravier et al. 2022b). Specifically, histone modifi- cations play a dynamic role in shaping the structure of chro- matin, influencing cellular transcriptional capacity (Tsuchida et al. 2017). On the other hand, DNA methylation levels of promoter regions (i.e., regions located upstream of transcrip- tional start sites) are generally associated with gene silencing in humans (Kangaspeska et al. 2008). Although this rela- tionship is not systematic, it is more frequently observed in promoters with (i) high CpG density, (ii) wide differentially methylated regions, or (iii) greater percentage methylation gains (de Mendoza et al. 2022). Furthermore, genes with methylation-repressed activity have a higher number of dis- tal regulatory elements (de Mendoza et al. 2022). Never- theless, positive correlations between promoter methylation and transcriptional changes are also found, which can be attributed to increased transcription factor affinity (Héberlé and Bardet 2019; Vizoso and van Rheenen 2021). Impor- tantly, the methylome of human skeletal muscle presents an epigenetic memory. For instance, the differentially methyl- ated sites remained unchanged during a detraining period of seven weeks, during which muscle mass returned to baseline (Seaborne et al. 2018). Strikingly, during a retraining period, greater hypertrophy was observed concomitantly with a greater abundance of hypomethylated genes involved in skeletal muscle growth (Seaborne et al. 2018). Recent stud- ies have also highlighted the crucial function of miRNAs in both acute responses and long-term adaptations to exercise by impairing the translation of their target mRNAs (Solsona and Sanchez 2021; Solsona et al. 2022). Furthermore, in recent years, there has been a growing interest in investigating the efficacy of thermal exposure to promote skeletal muscle regeneration following injury and attenuate atrophy. Atrophy can be defined as a signifi- cant loss of muscle mass and can result from immobiliza- tion during injury (Wall et al. 2013). Acutely, heating the ambient air of C2C12 cultured skeletal myotubes (47 °C for 30 min) modulated the pathways involved in protein turno- ver, including protein folding, pre-messenger RNA process- ing, messenger RNA splicing, and proteolysis (Szustakowski et al. 2007). Furthermore, the muscle atrophy F-box gene (MAFbx/Atrogin-1), an ubiquitin E3 ligase involved in pro- teasomal degradation, was upregulated in response to acute heat shock in mice myotubes (Szustakowski et al. 2007). Of note, these responses normalized within 24 h after acute heat stress, restoring cellular growth conditions after heat shock.

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281 European Journal of Applied Physiology (2025) 125:277–301 The autophagy machinery, involving forkhead box class O proteins (FOXOs) and autophagic genes (Atgs), is a criti- cal pathway for cell homeostasis and muscle integrity under stress (Sanchez et al. 2014; Møller et al. 2019). Autophagy is responsible for the degradation of proteins, lipids, pol- ysaccharides, and intracellular organelles like damaged mitochondria (Sanchez et al. 2012b, 2014, 2015). Thus, autophagy plays a major role in skeletal muscle atrophy, particularly in the context of immobilization due to injury. In elderly individuals or patients with chronic pathologies (e.g., sarcopenia, cachexia), autophagy promotes atrophy, which can cause oxidative stress, impaired mitochondrial function, myofiber denervation, weakness, and premature mortality (Castets et al. 2013; Carnio et al. 2014; Solsona et al. 2021). Consequently, investigating the effects of ther- mal exposure on atrophy and skeletal muscle regeneration, with a special focus on the involvement of autophagy, has become an emerging area of research. Hence, this narrative review aims to provide updated find- ings on the effects of thermal interventions on long-term exercise training adaptations for both endurance and resist- ance training. The second aim is to present the physiologi- cal and molecular effects of thermal interventions during skeletal muscle regeneration and discuss the potential use of thermal interventions in the context of muscle atrophy induced by immobilization. Moreover, the modulation of the autophagy pathway induced by thermal interventions dur- ing limb immobilization is detailed. Additionally, the review examines perspectives on the physiological adaptations induced by thermal interventions, particularly in relation to epigenetic modifications. Special attention will be given to the possible involvement of epigenetic factors, such as DNA methylation, in adaptation to heat intervention. Finally, the physiological effects of heat and cold interventions will be described, with a focus on the cellular responses involved in regulating skeletal muscle mass, including the role of the MTORC1 pathway in mRNA translation to proteins, ribo- some biogenesis, and satellite cell recruitment. Effects of repeated thermal interventions on adaptations to training The impact of adding cold interventions to exercise training regimes The effects of repeated cold exposures on long-term adap- tations to training have been of growing interest in the last decade. A study conducted during a 14-day training camp observed a lower increase in creatine kinase (CK) activity compared to the control group in the athletes who under- went twice-daily WBC sessions (3 min at −120 °C for seven consecutive days) (Zembron-Lacny et al. 2020). However, the increase in serum myoglobin was not different between groups. On the other hand, the oxi-inflammatory mediators (i.e., the levels of hydrogen peroxide (H2O2), nitric oxide (NO), interleukin-1β, C-reactive protein, hepatocyte growth factor, insulin-like growth factor, platelet-derived growth factor and vascular endothelial growth factor) were higher in the control group. It is well-established that there is a strong correlation between accumulating reactive oxygen species (ROS) and the inflammatory response (Mittal et al. 2014). Cryotherapy may reduce this response, thereby attenuat- ing the oxidative stress induced by exercise training. It is important to note that both inflammation and pro-oxidative imbalance are necessary for athletes to achieve adaptation (Mackey et al. 2007). A study investigating the effects of repeated WBC (3 min at −110 °C, 5 days/week for 2 weeks) on body composition found significant reductions in body fat percentage, visceral fat area and fasting glucose levels in healthy participants (Kozłowska et al. 2021). Furthermore, the study reported a reduction of circulating insulin and the homeostatic model assessment for insulin resistance score in middle-aged participants (∼ 50 years old). These improve- ments were partly attributed to their higher baseline values compared to their younger counterparts. Previous studies have provided evidence that cold inter- ventions can inhibit certain adaptations to exercise train- ing. CWI has been shown to decrease the activity of the MTORC1 signaling pathway (Roberts et al. 2015b), which is essential for protein translation, cell growth and muscle hypertrophy (Sanchez et al. 2019). CWI also reduces ribo- some biogenesis and attenuates exercise-related changes in the satellite cell pool (Roberts et al. 2015b; Figueiredo et al. 2016). On the other hand, CWI blunts and delays the increase in circulating testosterone and cytokines such as IL-6 and tumor necrosis factor-alpha (TNF-alpha) after resistance exercise (Earp et al. 2019). CWI has been shown to hamper various training adaptations following strength training, with significant effects on satellite cell activation and the activity of proteins that regulate muscle growth after a single bout of resistance exercise (Roberts et al. 2015b; Méline et al. 2017). Over a long period, CWI can lead to fewer gains in hypertrophy and completely blunt maximal isometric strength gains within a training protocol compris- ing 12 weeks of biweekly resistance training (Roberts et al. 2015b). In this study, CWI impeded the training-induced increase in the CSA of type II fibers, as well as the increase in the number of myonuclei per fiber. Therefore, caution must be taken when using regular cryotherapy during resist- ance training interventions. Regarding endurance exercise training, results appear more contrasted. First, although CWI may increase gene expression of biogenesis mitochondrial markers (e.g., per- oxisome proliferator-activated receptor-gamma coactiva- tor-1alpha (PGC-1alpha)) after a single endurance exercise

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282 European Journal of Applied Physiology (2025) 125:277–301 session, no effect was found in protein content of these mark- ers on long-term studies (Broatch et al. 2018). Importantly, a six-week sprint interval training protocol with post-exer- cise CWI at 10 °C for 15 min was not detrimental for peak aerobic power improvement, maximal oxygen consumption ( ̇VO2max) , maximal uncoupled respiration of mitochon- drial complexes I and II, or time trial performance (Broatch et al. 2017). A study showed that four weeks of CWI after high-intensity interval training did not alter endurance per- formance gains in healthy subjects (Aguiar et al. 2016). Moreover, a recent study revealed that repeated WBC (30 s at −60 °C, 150 s at −120 °C, 2 times/week, 6 weeks) did not adversely affect markers of endurance or resistance perfor- mance, such as ̇VO2max or muscle torque (Haq et al. 2022). However, jump height seems to be affected by WBC (Haq et al. 2022). The authors conclude that WBC does not blunt adaptations of endurance and resistance training, even if there may be an interference effect in the development of explosive power. Altogether, these findings suggests that CWI does not blunt adaptations to endurance training. On the other hand, multiple WBC sessions were found to reduce ROS and nitrogen oxide species (NOS) in blood samples after intense period of training including endurance, directed training and strength exercises in wrestle athletes (Zembron- Lacny et al. 2020), potentially leading to a reduced regenera- tive response. Indeed, the concentration of growth factors appeared to be negatively correlated with ROS and NOS content in the WBC group (Zembron-Lacny et al. 2020). Despite these findings, unilateral CWI blunted the gains in time to exhaustion during a single-leg incremental exercise in sedentary subjects who trained four times per week during four weeks. Furthermore, the percent change in ̇VO2max was significantly higher in the control leg (+ 8.2%) compared to the CWI-treated leg (−2.2%). After six weeks of training on different subjects, both groups showed a similar increase in time to exhaustion, but ̇VO2max and the ventilatory thresh- old were enhanced in the control leg only (Yamane et al. 2006). This suggest that CWI blunts adaptations to strength- endurance training. In summary, current research suggests that cryotherapy may interfere with certain training adaptations. To date, at least five studies have provided strong evidence that repeated exposures to cold interventions following resistance exercise hinder training adaptations (i.e., hypertrophy and isometric strength) by inhibiting the MTORC1 pathway, impairing ribosome biogenesis and muscle protein synthesis rates, and altering satellite cell dynamics (Yamane et al. 2006; Roberts et al. 2015b; Figueiredo et al. 2016; Fyfe et al. 2019; Fuchs et al. 2020) (Fig. 1e). However, cryotherapy appears less detrimental to endurance training adapta- tions, excepted for strength-endurance where CWI appears to blunt gains in performance (Malta et al. 2021). Finally, given that literature remains elusive on the effects of CWI on endurance performance, especially in low-intensity and high-volume training, further research is needed to clarify the mixed results. The studies included in this section and treating about the effects of repeated cold interventions on adaptations to training and muscle function are summarized in Table 1. The impact of adding heat interventions to exercise training regimes Recent evidence suggests that heat interventions can signifi- cantly enhance some long-term training adaptations. Regard- ing adaptations to endurance training, heat exposures (40 °C heated chamber, 30 min/day, 5 days/week for 3 weeks) may additionally enhance endurance-induced mitochondrial adaptations in rodents (Tamura et al. 2014). Indeed, post- exercise heat treatment increases mitochondrial enzyme activity (citrate synthase) and respiratory chain protein content in skeletal muscle, resulting in improved mitochon- drial function (Tamura et al. 2014). In humans, it has been revealed that short-wave diathermy can improve mitochon- drial respiratory capacity to a similar magnitude as observed with exercise (Marchant et al. 2022). However, in the latter study, only exercise significantly increased fatty acid oxi- dation and citrate synthase activity (Marchant et al. 2022). Repeated moderate heat exposure (warm baths, ~ 40 °C for 30 min, 5 days/week, 4 weeks) may benefit the cardiovas- cular system by decreasing resting sympathetic activity and heart rate, reportedly without altering baroreflex sensitiv- ity and sympathetic responses to stress (Cui et al. 2022). Moreover, passive heat interventions (40 °C heated cham- ber, 40–50 min/day, 3 days/week for 6 weeks) can increase skeletal muscle capillarization and endothelial NO synthase content similarly to moderate-intensity continuous training in young sedentary males (Hesketh et al. 2019). Accord- ingly, previous findings from Scoon and colleagues high- lighted that post-exercise sauna bathing (~ 30 min/day, ~ 4 days/week for 3 weeks, ~ 90 °C, humid sauna) may also be beneficial for increasing blood volume and endurance per- formance in sub-elite athletes (Scoon et al. 2007) (Fig. 1 g). The authors of this study conclude that repeated post-exer- cise sauna bathing produces a worthwhile improvement in endurance athletes. They report a 32% increase in the time to exhaustion test, which is estimated to correspond to a 1.9% enhancement in performance over a 5-km time trial. Alto- gether, these results indicate that heat interventions could be an effective approach for improving muscle function and enhancing exercise performance in sedentary population and sub-elite endurance athletes, respectively. However, a recent study failed to find effects of post-exercise dry sauna bathing (30 min, 3 times per week for 4 weeks, 89 ± 3 °C, 10 ± 2% relative humidity) on hematological adaptations in physical education students (Sitkowski et al. 2022). Indeed,

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283 European Journal of Applied Physiology (2025) 125:277–301 Table 1 Effects of repeated cold intervention on adaptations to training and muscle function 1RM one repetition maximum, AMPK AMP-activated protein kinase, CWI cold water immersion, ET endurance training, HIIT high-intensity interval training, HSP heat shock protein, MAPK mitogen-activated protein kinase, p38 p38 mitogen-activated protein kinases, RT resistance training, SIT sprint interval training, ̇VO2max) maximal oxygen consumption, WBC whole-body cryotherapy Study Main effects Exercise Population and samples Recovery method Effects Yamane et al. 2006 No increase in maximal oxygen uptake and ventilatory threshold in the CWI group ET Healthy students CWI – 5 min of cycling at 35% ̇VO2max) followed by 25 min at 70% ̇VO2max 2*20 min; 4 days/week; 4 weeks; 5 ± 1 °C Yamane et al. 2006 No increase in muscle endurance capacity and lower increase in maximal muscle strength RT Healthy students CWI – 3 × 8 handgrip exercises at 70–80% 1RM, with 160 s of rest 20 min; 3 days/week; 4 weeks; 10 ± 1 °C Roberts et al. 2015a, b Lower increase in hypertrophy and strength Strength training Physically active men CWI – Blunt the activation of satellite cells Vastus lateralis biopsies 10 min; 2 days/week; 12 weeks; 10.1 ± 0.3 °C Aguiar et al. 2016 No differences in endurance performance gains compared to the control group HIIT Healthy subjects CWI ∅ No differences in protein expression com- pared in protein expression compared to the control group (Hsp72, AMPK, p38 MAPK) 8–12 cycling 60 s at 90–110% of peak power followed by 75 s of active recovery Vastus lateralis biopsies 15 min; 3 days/week; 4 weeks; 10 °C Broatch et al. 2017 Not detrimental to endurance adaptations (peak aerobic power, maximal oxygen consumption, time trial performance) SIT Physically active participants CWI ∅ 5-min cycling at 75 W, followed by 4–6 × 30-s “all-out” efforts Vastus lateralis biopsies 15 min; 3 days/week; 6 weeks; 10 °C Fyfe et al. 2019 Lower increase in hypertrophy RT Recreationally active men CWI – Different exercises on lower and upper limbs; training intensity set at 12-RM and 120 s of recovery between each exercise Vastus lateralis biopsies 15 min; 3 days/week; 7 weeks; 10ºC Fuchs et al. 2020 Reduction in myofibrillar protein synthesis rates RT Healthy young men CWI – 4 × 8–10 repetitions on leg press and knee extension machines; training intensity set at 80% 1-RM Vastus lateralis biopsies and blood samples 20 min; 3–4 days/week; 2 weeks; 8ºC Zembron-Lacny et al. 2020 Lower increase in creatine kinase activity Training camp: endurance training (50%), directed training (24%) and special power training (26%) Elite athletes WBC + Blood samples Twice daily for seven consecutive days; 3 min at −120 °C Kozłowska et al. 2021 Decrease in body fat percentage, visceral fat area, fasting glucose and circulating insulin Only cooling Healthy participants WBC 3 min; 5 days/week for 2 weeks; −110 °C + Blood samples Haq et al. 2022 No differences in endurance and resistance performance gains compared to the control group RT (strength and plyometric training ses- sions) and ET (30 min of running at 70% ̇VO2max) ) Healthy male participants WBC 3 min; 2 days/week for 6 weeks; 30 s at −60 °C and 150 s at −120 °C ∅ No increase in jump height

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284 European Journal of Applied Physiology (2025) 125:277–301 no additional effects were found on hematological variables (i.e., hemoglobin mass, ferritin concentration) and exercise capacity (i.e., peak oxygen uptake, maximal power and power at gas exchange threshold) with heat interventions. This conflicting result might be explained by the differ- ences in humidity (humid versus dry sauna). A humid sauna increases more the thermal load than a dry sauna and might explain the differences observed in hematological adapta- tions and exercise performance (Henderson et al. 2021). It is also suggested that a higher temperature (∼100 °C) might be necessary to have positive effects on exercise per- formance. In this sense, a recent study has demonstrated that dry sauna bathing at 100 ± 3 °C (40 min, 3 days/week, 3 weeks) increases physical performance (work capacity and muscle flexibility) in semi-professional football players (Bartolomé et al. 2021). Despite these findings, studies on the effects of heat interventions on training adaptations in other disciplines, particularly high-intensity sports, remain limited. It is worth noting that, in short-track speed skat- ing, a discipline associating explosive power and complex coordination, maximal isometric strength of the knee exten- sors can be increased by the regular use of HWI (40.3 °C for 20 min, 4 days/week for 4 weeks) (Méline et al. 2021). In addition, a tendency was observed for improvement in ̇VO2max in these elite athletes (Méline et al. 2021). However, it was also recently found that regular HWI may impair some adaptations in elite teen archers, another discipline requir- ing complex coordination but less mechanical and energetic constraints than short-track speed skating (Hung et al. 2018). Indeed, it was found that shooting performance and pos- tural stability decreased after HWI (30 min at 40 °C twice a week for two weeks) (Hung et al. 2018). This result suggests that caution should be exercised when considering HWI for activities requiring high control of postural stability. Furthermore, results appear more controversial concern- ing the effects of heat interventions on muscle mass and strength (Labidi et al. 2021). It was previously observed in healthy subjects that repeated and prolonged localised heat exposures (8 h/day, 4 days/week during 10 weeks) through heat- and steam-generating sheets can increase maximal iso- metric strength by 5.8%, muscle size by 2.7–6.1% and fiber CSA by 8.3%, without exercise training (Goto et al. 2011). Moreover, Hafen and colleagues investigated the effects of six days of localised heat interventions (pulsed shortwave diathermy, 2 h/day for 6 consecutive days) (Hafen et al. 2018). They found an increased expression of mitochondrial biogenesis markers (PGC-1alpha, METP (mitochondrial electron transport protein) complexes I and V). Hence, mus- cle heating seems to stimulate signaling pathways involved in muscle function (i.e., protein synthesis and mitochondrial biogenesis). Strength and muscle contractile function are also increased with repeated heat exposures (Racinais et al. 2017; Kim et al. 2020). Interestingly, there may be a negative correlation between training status and the physiological effects of heat interventions, with elite athletes deriving fewer benefits compared to sedentary or people affected by disease. A recent study investigated the effects of six weeks of localised heat interventions (heat pads, 8 h/day, 5 days/ week for 6 weeks) on muscle oxidative and microvascular function in physically active participants (Ihsan et al. 2023). There were no effects on gastrocnemius aerobic function. This study supports the hypothesis that trained subjects require a more potent heat stimulus (increased duration and/ or temperature) to undergo skeletal muscle adaptations. A recent study seems to corroborate this hypothesis, showing no effects of post-resistance exercise HWI on body compo- sition and resistance performance in rugby players (Horgan et al. 2023). Moreover, the differences in muscle temperature across studies could potentially account for discrepancies in the physiological benefits observed with heat interventions. In this sense, a recent study emphasizes the need to achieve a potent stimulus by elevating muscle temperature above 39 °C to induce significant physiological adaptations (Ihsan et al. 2023). Altogether, these data highlighted that repeated heat interventions may enhance adaptations to endurance and resistance training (i.e., high-intensity endurance per- formance and isometric strength) when muscle temperature is sufficiently increased. Thus, it is of paramount importance to measure and ensure that the muscle temperature reaches 39 °C. Finally, caution must be taken regarding the optimal temperature and exposure duration when using heat inter- vention. Indeed, a recent study compared the impact of two different HWI interventions (45 min at 40 °C versus 41 °C) immediately after exercise-induced muscle damage (EIMD, five sets of 10 maximal eccentric contractions) on core body temperature and functional markers of muscle recovery (Sautillet et al. 2023). The authors highlighted that 45 min of HWI at 41 °C, elevating core temperature to 38.5–39 °C by the end of the immersion, may be the opti- mal dose to improve muscle recovery in the late recovery phase (24–48 h post-exercise). Specifically, HWI at 41 °C reduced the EIMD-induced decline in the rate of force development and concentric peak torque (at 24 and 48 h, respectively) (Fig. 1d). Hence, this study shows that there is a strong dose–response relationship for heat intervention, and that a difference of 1 °C during HWI can lead to differ- ent results. Another study found that 2 h of HWI at 42 °C increases muscle temperature up to 39 °C, with no increase in systolic blood pressure. The authors concluded this pro- tocol is safe and tolerable, and that the increase of 2.8 °C in muscle temperature corresponds to the optimal zone for promoting cellular adaptations and muscle growth (Rodri- gues et al. 2020a). However, while transient heat stress may benefit skeletal muscle adaptations, prolonged heat inter- vention could dysregulate cellular homeostasis and become

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285 European Journal of Applied Physiology (2025) 125:277–301 detrimental. Accordingly, a study conducted in pigs showed that 24–72 h of environmental heat stress (35 °C) signifi- cantly changed the expression of markers of phagophore formation and maturation and autophagosome degradation (UNC-51-like kinase 1 phosphorylation, Microtubule-asso- ciated protein light chain 3 A/B-II to A/B-I ratio, p62 pro- tein expression), indicating an accumulation of autophago- somes in muscle cells (Brownstein et al. 2017). Although the autophagic flux were not directly measured, these results may reflect decreased lysosomal degradation. Of note, PINK1 and BNIP3L (PTEN‐induced putative kinase 1 and BCL2/Adenovirus E1B 19 kDa interacting protein 3‐like), two mitophagic markers, decreased with heat exposure. This would imply impaired renewal of cellular components, par- ticularly mitochondria, raising concerns about the viability of such prolonged treatments for maintaining cellular quality and function. In summary, improper use of thermal interventions can delay recovery and impair training adaptations, such as gains in isometric and endurance strength, and hypertrophy, as observed with the regular use of CWI (Takagi et al. 2011; Roberts et al. 2015b; Méline et al. 2017; Malta et al. 2021). Therefore, matching specific training stimuli with the appro- priate use of thermal strategies is fundamental to avoid inter- ferences and to potentially favour training adaptations. While cryotherapy has been shown to blunt certain adaptations to training, only a limited number of studies have investigated the effects of repeated heat interventions on adaptations to exercise. However, recent data suggest that heat interven- tions may increase isometric strength and endurance per- formance, without changes in muscle mass, in trained ath- letes (Scoon et al. 2007; Méline et al. 2021) (Fig. 1f). The impact of passive heat treatment on skeletal muscle mass and properties in healthy participants remains to be clarified. In addition, further studies are needed to establish a stronger consensus on the effects of repeated thermal interventions on sport-specific tasks. For example, little is known about the impact of the regular use of heat intervention on repeated sprint ability in team sports or specific performance in disci- plines requiring both endurance and strength qualities (e.g., artistic gymnastics). On the other hand, because of the multi- factorial nature of recovery, which also includes psychologi- cal components, it is crucial to consider individual prefer- ences (Minett and Costello 2015). Finally, literature does not allow to make definitive statements about the impact of heat interventions on skeletal muscle mass and properties. Fur- ther studies are needed in humans to determine the optimal heat dose (e.g., temperature and duration). A dose–response relationship seems to exist, and other stimuli must be consid- ered, such as the hydration level of the participants following the intervention, their training level, the kind of intervention (sauna-bathing, localized heating, hammam, etc.), and fat mass percentage. The studies included in this section and discussing about the effects of repeated heat intervention on adaptations to training and muscle function are summa- rized in Table 2. Figure 1 details the effects of cold and heat interventions on training adaptations and muscle function. Exercise and thermal interventions alter epigenetic factors: current evidence and perspectives Recent investigations have revealed that endurance exercise provoked global and gene-specific changes in the methyla- tion levels in skeletal muscle biopsies of healthy sedentary men (Barrès et al. 2012). Concretely, the authors observed reduced methylation at the promoters of several genes involved in cellular metabolism: PGC-1alpha, PDK4 (pyru- vate dehydrogenase kinase 4), and PPAR-delta (peroxisome proliferator-activated receptor-delta), along with increased mRNA abundance. This suggests that changes in the meth- ylation level of these genes are correlated with mRNA expression. Importantly, these results depended on exer- cise intensity, with stronger hypomethylation and expres- sion modifications after high-intensity exercise. Training intervention also impacts DNA methylation during endur- ance exercise, as shown by a study conducted on humans with unilateral endurance tasks (Lindholm et al. 2014). However, these modifications mostly concerned enhanc- ers, gene bodies and intergenic regions. These data suggest that while promoters may be involved in quick adjustments, other regions are likely involved in long-term adaptations to exercise training. In this sense, twelve weeks of resistance and high-intensity interval training led to minor changes in the methylation of DNA promoter regions (Robinson et al. 2017). A recent study conducted in our laboratory showed that global methylation increases after a single sprint inter- val training (Solsona et al. 2024). Furthermore, the meth- ylation of an enhancer of the gene-inducible NO synthase increased after exercise, suggesting that exercise is a potent stimulus to induce epigenetic modifications. Importantly, the magnitude of muscle deoxygenation during exercise was associated with the changes in DNA methylation of both the promoter and the enhancer of this gene (the relationship was negative for the enhancer). Furthermore, average heart rate was also correlated with the modifications of the promoter methylation. These results offer insight into the potential link between the stress induced by a sprint interval session and alterations in methylation patterns. Further research is necessary to determine if these modifications influence gene expression. The relationship between resistance exercise and DNA methylation has also been explored, although the number of studies in this domain remains limited. Nonetheless, these studies have provided valuable insights into the fluctuations in DNA methylation and their connection to gene expression (Seaborne and Sharples 2020). For instance, research has

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286 European Journal of Applied Physiology (2025) 125:277–301 Table 2 Effects of repeated heat intervention on adaptations to training and muscle function Study and design Main effects Exercise Population and samples Recovery method Effects Scoon et al. 2007 Increased run time to exhaustion and plasma volume Regular training Competitive male runners Sauna + ~ 30 min/day; ~ 4 days/week for 3 weeks; ~ 90°C Goto et al. 2011 CSA expansion Only heating Healthy men Heat and steam sheets ∅/ + Increased MVC of the knee extensors (5.8%) but not the flexors Vastus lateralis biopsies 8 h/day; 4 days/week for 10 weeks; muscle temperature maintained at 38.3°C Tamura et al. 2014 Increased p38 MAPK and p70S6K phosphorylation ET Mices (6-week-old males) Hot chamber + Decreased AMPK and ACC phospho- rylation Plantaris and soleus muscles 30 min/day; 5 days/week for 3 weeks; 40°C Increased mitochondrial enzymes activity and respiratory chain protein content 30 min of treadmill running 5 days/ week for 3 weeks at 25 m/min Racinais et al. 2017 Improvement of muscle contractile function with an increase of the peak twitch amplitude and the maximal torque production Only heating Male participants Environmental chamber + 1 h/day for 11 consecutive days; 48–50 °C at 50% RH Hafen et al. 2018 Increase of HSP70, HSP90 and PGC- 1alpha expression Only heating Healthy sedentary volunteers Pulsed shortwave diathermy + Increase of mitochondrial electron transport protein complexes I and V expression Vastus lateralis biopsies 2 h/day for 6 consecutive days; muscle temperature increased by ~ 3.9 °C Hung et al. 2018 Decreased shooting performance, pos- tural stability and DHEA-S levels Regular training Elite teen archers HWI ∅/- No effect on motor unit recruitment, autonomic system activity or cortisol levels Saliva 30 min/day; 2 days/week for 2 weeks; 40°C Hesketh et al. 2019 Increase in skeletal muscle capillariza- tion and eNOS content ET Young sedentary males Hot chamber ∅/ + Increase in skeletal muscle mitochon- drial density, GLUT4, or IMTG content in the exercise group only 40–50 min of cycling on a cycle ergom- eter at an intensity eliciting ~ 65% V̇O2peak Vastus lateralis biopsies 40–50 min/day; 3 days/week for 6 weeks; 40 °C Kim et al. 2020 Increase in eNOS content Only heating Healthy young adults Garment perfused with water at ~ 52 °C + Increase the strength of the knee exten- sors Vastus lateralis biopsies 90 min/day; 5 days/week; 8 weeks; muscle temperature increased to ~ 37 °C Bartolomé et al. 2021 Increase in physical performance (work capacity and muscle flexibility) Regular training Semi-professionnal football players Dry sauna + 40 min/day; 3 days/week; 3 weeks, 100 ± 3 °C

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287 European Journal of Applied Physiology (2025) 125:277–301 Table 2 (continued) Study and design Main effects Exercise Population and samples Recovery method Effects Labidi et al. 2021 No increase in CSA and in strength Only heating Active participants Heat pads ∅ 8 h/day; 5 days/week; 6 weeks; muscle temperature increased by ~ 4.6 °C Méline et al. 2021 Increase in maximal isometric strength of the knee extensors Regular training Elite short-track speed skaters HWI ∅/ + Increase trend in V̇O2max 20 min/day; 4 days/week for 4 weeks; 40.3 °C Cui et al. 2022 Decreased resting sympathetic activity and heart rate Only heating Healthy older volunteers HWI + 30 min/day; 5 days/week for 4 weeks; ∼40 °C Marchant et al. 2022 Increase in mitochondrial respiratory capacity RT (4 × 4 min of single-leg extension at 80% of work rate max with 4 min of active recovery at 20% of work rate max) Sedentary young subjects Short-wave diathermy ∅/ + Fat oxidation capacity, citrate syn- thase activity and exercise capacity increased in the exercise group only Quadriceps femoris biopsies 60 min/day; 3 days/week for 6 weeks; muscle temperature elevated at 39.5°C Sitkowski et al. 2022 No additional effects on hematological variables and on exercise capacity ET Physical education students Sauna bathing ∅ 60 min stationary cycling Blood samples 30 min/day; 3 days/week for 4 weeks; 89 ± 3 °C, 10 ± 2% RH Horgan et al. 2023 No increase in lean muscle mass and in resistance performance Heat-RT Rugby players HWI ∅ 15min/day; 2 days/week por 4 weeks; 39.3°C Ihsan et al. 2023 No increase in gastrocnemius aerobic function Only heating Physically active participants Heat pads ∅ 8 h/day; 5 days/week for 6 weeks; mus- cle temperature elevated at 37.6°C ACC acetyl-coenzymeA carboxylase, CS citrate synthase, CSA cross-sectional area, DHEA-S dehydroepiandrosterone sulfate, eNOS endothelial nitric oxide synthase, ET endurance training, GLUT4 glucose transporter type 4, HAD 3-hydroxyacyl coenzymeA dehydrogenase, HSP heat shock protein, HWI hot water immersion, IGF-1 insulin-like growth factor 1, IGF2R insulin-like growth factor2 receptor, IMTG intramuscular triglyceride, [La−] blood lactate concentration, MVC maximal voluntary contraction, p70S6K ribosomal protein70 S6 kinase, PGC-1alpha peroxi- some proliferator-activated receptor-gamma coactivator-1alpha, PHF20 plant homeodomain finger protein 2, RT resistance training, TNIK Traf2- and Nck-interacting kinase, TFDP2 transcrip- tion factor, Dp-2/E2F dimerization partner 2, TTN titin, UQCRB ubiquinol-cytochrome c reductase binding protein, V̇O2peak peak oxygen uptake

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288 European Journal of Applied Physiology (2025) 125:277–301 shown that resistance training elicits differential methylation and messenger RNA expression changes of growth factors such as growth hormone-releasing hormone and fibroblast growth factor-1 in the leukocytes of young men (Denham et al. 2016). On the other hand, acute resistance exercise can modulate global methylation as evidenced by the hypometh- ylation of long interspersed nuclear element-1 in resistance- trained, but not in sedentary subjects (Bagley et al. 2020). Interestingly, it was found that metabolic genes such as glycerol-3-phosphate acyltransferase (GPAM) and sterol regulatory element binding transcription factor 2 (SREBF2) exhibited hypermethylation in accustomed participants. At the same time, the methylation levels decreased for SREBF2 in the inexperienced group (Bagley et al. 2020). Thus, oppo- site responses were obtained for SREBF2 depending on the training status. These contrasting findings could be attrib- uted to the fact that resistance training represents a meta- bolic stress for sedentary people, whereas trained subjects may experience less disruption to metabolic homeostasis. Given the inverse association between gene methylation and expression levels (Cortés-Mancera et al. 2022), this result suggests that SREBF2 was expressed in the unexperienced group and inactivated in resistance-trained individuals, even though SREBF2 mRNA levels were not measured in the study. Therefore, epigenetic responses may largely depend on the fitness status, probably because it can modulate the stress response to a given type of exercise. Surprisingly, the methylation levels of genes associated with inflammation (IL-6 and TNF-alpha) or hypertrophy (MTOR and Akt) were not affected in either group. Furthermore, in the context of exercise training, epigenetic modifications also rely on the nutritional status. In a crossover study, trained cyclists per- formed a high-intensity interval exercise (8 × 5 min at 82.5% of peak power output) in the evening before being split into two groups: fasted and fed (Lane et al. 2015). The next morning, they all completed 2 h of cycling at 50% of peak power output before being fed with a standardized meal. The results of the study show that 4 h post-exercise, the promoter of PPAR-delta exhibited hypermethylation in the overnight fasting condition only. It has been established that environmental conditions affect cellular function through epigenetic mechanisms. Importantly, different heat exposure durations have diverse effects on the epigenome (Malabarba et al. 2021). Hot condi- tions regulate miRNAs, single interfering RNAs, long non- coding RNAs, histone variants, chaperones and covalent modifications, chromatin remodeling and DNA methylation in plants (Liu et al. 2015). These results are of paramount importance because they establish a wide range of epige- netic modifications in response to heat exposure. Similarly, miRNA-297 was upregulated during the heat acclimatization period in rats (Tetievsky et al. 2014), showing that altera- tions in the epigenome occur in mammals with thermal interventions. In cultured mouse skeletal myoblasts, thermal stress also influences epigenetics, as shown in a study that examined the impact of different temperatures on histone modifications (Sajjanar et al. 2019). In this study, the rela- tive expression of the histone acetyltransferase Gcn5 and histone H4-acetylation increased at 41 °C. In high-temper- ature conditions, the relative mRNA level of the de novo DNA methyltransferase 3A (DNMT3A) increased, while it decreased in cold conditions. Contrastingly, the methyl- transferase responsible for the maintenance of methylation (DNMT1) increased in the cold conditions (Sajjanar et al. 2019). Global DNA methylation responded differently in the heat versus cold conditions, with a higher amount of 5-methylcytosine at 39 °C compared to 35 °C. Interestingly, heat stress reduced maximal respiration and spare respira- tory capacity, suggesting impaired mitochondrial function in C2C12 myoblasts. Conversely, cold stress stimulated glycol- ysis as a thermogenic response to meet energy requirements. Lastly, glycolytic capacity increased in all conditions com- pared to the control. Taken together, these results indicate an interplay between energy metabolism and epigenetics during cell stress response (Sajjanar et al. 2019). As acetyl and methyl groups are covalent modifications, they appear to be thermodynamically stable (Delatte and Fuks 2013). Thus, although the mechanisms behind the modulation of epigenetic modifications are not fully understood, these data seem to indicate that hot and cold conditions differ- ently affect enzymes that regulate epigenetic marks and the subsequent adaptations to stress. The effects of thermal interventions on ten-eleven trans- location (TET) proteins (i.e., DNA demethylases) should be assessed for a deeper understanding of the regulation of DNA methylation. Importantly, it has been shown that physical activity prevents the decreased expression of TET proteins, as shown in the hippocampus of aged mice (Jessop and Toledo-Rodriguez 2018). Consequently, the promoter region of miRNA-137 was rich in 5-hydroxymethylcytosine, suggesting enhanced neurogenesis and improved memory. However, this result was not found in the hypothalamus, indicating distinct regulation processes according to the cer- ebral zones. On the opposite, exercise training hypermethyl- ated genes related to hypertension while TET1 expression decreased in hypertensive rats, contributing to enhanced mesenteric arterial function and vasodilation (Zhang et al. 2020). Another study showed that heat-induced global hypo- methylation in microspores and the amount of differentially methylated genes were temperature-dependent (Li et al. 2016), evidencing a dose–response relationship. Corre- spondingly, thermal stress reduced the levels of DNA meth- ylation in corals, which hypothetically leads to transcrip- tional plasticity (Dimond and Roberts 2016). This means that heat exposure could open a window of opportunity for enhancing training adaptations. Another study assessed the

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289 European Journal of Applied Physiology (2025) 125:277–301 impact of 21 days of heat exposure on epigenetics in pigs. The data showed that constant heat stress induced global hypermethylation, but several genes involved in muscle growth and development were hypomethylated. Myosin heavy chain 11, implicated in muscle fiber properties, was identified among the hypomethylated genes. Additionally, genes encoding glycolytic enzymes also displayed differ- ential methylation, leading to increased protein expression. For example, PDK3 was hypomethylated under constant heat conditions, resulting in increased expression. PDK3 plays a role in inhibiting the conversion of pyruvate to acetyl- coenzyme A, thereby inhibiting aerobic processes in skeletal muscle (Hao et al. 2016). Taken together, these data strongly suggest that thermal interventions affects the epigenome. Overall, the effects of exercise on epigenetics need fur- ther attention, especially in humans and in combination with thermal interventions. The influence of epigenetic modifica- tions on the expression of specific genes should be a grow- ing research axis in the next years. Indeed, it represents an essential perspective for both athletes and patients to better understand the effectiveness of exercise prescriptions on skeletal muscle adaptations. Of note, epigenetic responses are tissue- and species-specific (He et al. 2011; Weyrich et al. 2020), and consequently, studies on the effects of heat exposure on humans are needed, notably in skeletal muscle. Thermal interventions and skeletal muscle regeneration Despite its notable impact on pain perception, cold inter- vention appears to hinder the process of muscle regen- eration in numerous animal studies. Research on rats and mice revealed that cryotherapy delays recovery after injury (Takagi et al. 2011; Miyakawa et al. 2020; Kawashima et al. 2021; Miyazaki et al. 2023). Cryotherapy can reduce the inflammatory response following skeletal muscle injury (i.e., freezing injury of the tibialis anterior in rats) by decreas- ing the infiltration of pro-inflammatory macrophages and the concentration of several inflammatory markers (Vieira Ramos et al. 2016). As evidenced by another study in rats, a twenty-minute cooling intervention immediately succeeding trauma delayed skeletal muscle regeneration (Takagi et al. 2011). Specifically, during the primary reaction of degen- eration and the early stages of regeneration, the authors observed a delayed macrophage migration to the injured site. The proportion of centrally nucleated fibers was higher in the icing group 14 days after injury, suggesting a slower regeneration. Furthermore, the CSA of the regenerating fib- ers was larger in the non-icing group 28 days post-trauma. Notably, collagen deposition was significantly higher in the icing group both at 14 and 28 days after injury. Still in the context of muscle contusion injuries in rats, icing has been associated with delayed neutrophil and macrophage infil- tration (Singh et al. 2017). The authors showed a reduced/ delayed infiltration of macrophages, reduced expression of angiogenic factors, and lower vessel volume for the cold group. Moreover, it was recently found that icing decreases M1 macrophage accumulation after skeletal muscle injury in rats (i.e., electrically stimulated eccentric model), lead- ing to an incorrect degradation of injured muscle fibers (Kawashima et al. 2021; Miyazaki et al. 2023). As a result, delayed and impaired muscle regeneration was observed. Hence, the successive phases of muscle degeneration and short-term inflammation appear essential for subsequent proper regeneration (Takagi et al. 2011; Miyakawa et al. 2020). Upon muscle injury, calcium release may activate the calpain proteases, initiating the removal of myofibrillar fragments before proteasomes can degrade these damaged structures (Bartoli and Richard 2005). However, temperature is an important regulator of the enzymatic activity of cal- pains (Pomponio and Ertbjerg 2012). Additionally, inflam- mation might play a crucial signaling role in the initiation of the regenerative process. Cold intervention by blunting inflammation disrupts the normal progression of recovery and alters the regeneration cascade. However, a recent study in rats introduces a contrasting perspective. In comparison to previous studies, Nagata et al. used a milder crush injury model inducing a limited muscle injury with a myofiber necrosis fraction of approximately 4% of the myofibers. Icing treatment (3 sets of 30 min immediately after injury, each with 90-min intervals, and 24 h and 48 h after), decreased the accumulation of inducible NO synthase-expressing mac- rophages and the percentage of necrotic cells, and increased the number of activated satellite cells and CSA, suggesting an improvement in skeletal muscle regeneration (Nagata et al. 2023). Interestingly, the authors suggested that icing could be an appropriate solution for improving regeneration from limited muscle damage, but should be avoided in case of large-scale muscle necrosis. The extrapolation of the results of these studies to humans presents limitations. Indeed, only one of these studies used an eccentric-damaging model, prompting whether the dam- age induced by freezing or muscle crush injuries is compa- rable to EIMD. Moreover, due to the smaller size of rat legs, applying ice for 30 min provides a severe cooling effect, whereas the same dose in humans may not result in com- parable reductions in muscle temperature (Bleakley et al. 2012). In humans, cryotherapy decreases the expression of blood markers of skeletal muscle inflammation (i.e., interleukin-1 and C-reactive protein), as well as oxidative stress markers (H2O2 and NO) after a 14-day training camp (Zembron-Lacny et al. 2020). It has been suggested that the attenuation of these indirect markers of muscle stress might delay skeletal muscle regeneration (Zembron-Lacny et al. 2020). Indeed, a decrease in inflammation and oxidative

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290 European Journal of Applied Physiology (2025) 125:277–301 stress might generate dysfunctional autophagy in muscle fibers, leading to an incomplete degradation of damaged organelles, and resulting in a delayed or impaired regenera- tion of skeletal muscle (Sandri et al. 2013; Sanchez et al. 2019; Romanello and Sandri 2021). However, to date, there are no studies that have focused on the impact of cryotherapy following a skeletal muscle injury in humans, even if it is a commonly used practice in sports traumatology (Frery et al. 2023). Data on the effects of heat intervention during skeletal muscle regeneration have also been explored. In a recent study examining the autophagic response during atrophy in rodents with sectioned soleus and plantaris tendons, the repeated application of heat mitigated the reduction in fiber CSA (Hirunsai and Srikuea 2021). This study demon- strated the beneficial effect of heat application using ther- mal blankets (40.5–41.5 °C, 30 min/day for 7 days) on the autophagic pathway in rats, particularly in the context of tenotomy-induced muscle atrophy. Moreover, the observed atrophy was linked to an elevation in autophagic markers. Of note, autophagy represents an important system in the degradation and renewal of cellular constituents (Sanchez et al. 2019; Botella et al. 2023). During stressful situations such as exercise, heat exposure, hypoxia or fasting, double- membrane vesicles called autophagosomes capture cytoplas- mic components, including lipids, proteins, and organelles such as mitochondria and ribosomes to limit unnecessary energy expenditure (Sanchez et al. 2019). These vesicles are subsequently engulfed by lysosomes and are degraded along with their contents. Again, heat intervention effectively tar- gets and limits autophagic processes at eight and fourteen days of immobilization, suggesting its role in preserving cellular components (Hirunsai and Srikuea 2021) (Fig. 2f). Heat treatment (thermal blankets, 30 min/day, 40.5–41.5 °C, Heat therapy during atrophy MTORC1 PGC-1a HSPs Satellite cell recruitment Mitochondrial homeostasis Activation Inhibition Protein aggregation and denaturation Autophagy Collagen deposition and fibrosis lysosome degradation autophagosome engulfment Protein synthesis Increase in muscle mass and strength Protein and mitochondria stabilisation Influence of epigenetics LC3BII Cathepsin-L Protein synthesis a. b. c. d. e. f. Fig. 2 Muscle regeneration with heat intervention during atrophy. Heat intervention promotes protein synthesis through several mechanisms: a promotion of satellite cell proliferation and differentiation, b induction of HSPs, and c modulation of the MTORC1 pathway. Heat interven- tion enhances mitochondria homeostasis via the PGC-1alpha pathway (d), and limits skeletal muscle fibrosis, collagen deposition and pro- tein aggregation (e). Additionally, heat intervention reduces the activ- ity of the autophagosome-lysosome system (Cathepsin L.), including mitophagy (f). However, most of the studies are conducted in non- human models (i.e., rat and mouse models) and translation between species remains challenging. In this sense, more research in humans is needed to establish the optimal protocol for incorporating heat inter- vention into therapeutic approaches. Future research is necessary to further understand the involvement of epigenetic modifications on the effects of heat intervention in skeletal muscle regeneration. These adap- tations collectively contribute to the increases in skeletal muscle mass and isometric strength during muscle injury

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291 European Journal of Applied Physiology (2025) 125:277–301 7 days) was found to reduce the expression of the protein Cathepsin L in the atrophied soleus muscle of rats, suggest- ing a decrease in lysosomal activity (14 days post-tenotomy). Consistently, heat intervention significantly limits the loss of muscle mass during atrophy in rats (Goto et al. 2004; Hir- unsai and Srikuea 2021). Moreover, heat treatment (30 min/ day, 42 °C, 6 weeks) reduces muscle atrophy (evidenced by CSA measurements) and increases markers of protein syn- thesis (Akt, mTOR, heat-shock protein (HSP) 70) in diabetic rats (AlSabagh et al. 2022) (Fig. 2 c.). Heat pre-conditioning (20 min, 43 °C, 48 h before exercise) also increased HSPs expression and attenuated the increases in CK plasma con- centration induced by downhill running in rats (Touchberry et al. 2012). Heating skeletal muscle can thus prevent the overactivation of this catabolic system in oxidative and gly- colytic muscles. Additionally, heat exposure (thermal blan- kets, 30 min/day, 40.5–41.5 °C, 7 days) reduces necrosis and decreases the expression of the apoptotic protein TNF-alpha in tenotomized soleus muscles in rats (Hirunsai and Srikuea 2020). During atrophy, the infiltration of macrophages was reduced with heat interventions. Heat intervention can also limit fibrosis, decreasing collagen deposition (Fig. 2e) in injured rat soleus muscle (bupivacaine injection), whereas cold stress has the opposite effect (Shibaguchi et al. 2016). A recent review includes heat intervention as a potential therapeutic strategy for skeletal muscle atrophy (Huang et al. 2022). Specifically, heat treatment upregulates HSP72, HSP25 and downregulates REDD1 (regulated in develop- ment and DNA damage responses 1), preventing muscle wasting (Nonaka et al. 2015; Tsuchida et al. 2017). Moreo- ver, in rats with diabetes-induced atrophy, heat intervention attenuates the decrease in fiber CSA (Nonaka et al. 2015). Finally, a recent meta-analysis concludes that passive heat treatment can act as an exercise mimetic and can be used as an alternative to exercise to increase muscle mass and strength and attenuate subsequent atrophy (Rodrigues et al. 2020b). However, unlike physical exercise, heat treat- ment only provokes small changes in tissue metabolism (Kaluhiokalani et al. 2024). Localised passive heat therapy, applied intermittently over six weeks, induces similar gains in resistance artery function (i.e., peak blood flow) at rest compared to exercise training, but smaller gains during a knee extension test (Kaluhiokalani et al. 2024). Moreover, even if the increase in endothelial nitric oxide synthase expression was similar between the two conditions, only exercise training enhanced angiogenesis (Kaluhiokalani et al. 2024). Additionally, unlike whole-body hyperthermia, local hyperthermia does not increase ventilation, systemic aerobic metabolism and left ventricular systolic and diastolic functions, even if leg blood flow and cardiac output were increased (Watanabe et al. 2024). The authors suggest that local hyperthermia, with leg tissue temperature ≤ 38 °C, is not a sufficient stimulus to activate metabolism and central cardiorespiratory-mediated mechanisms. Hence, passive local heat treatment seems to induce fewer adaptations compared to exercise. Therefore, heat exposure should not be considered a substitute for exercise training but rather a complementary intervention. A recent study examined the effects of passive muscle heating (hot pack, 42 °C, 20 min) on skeletal muscle regen- eration after a crush injury on the extensor digitorum longus muscle in rats (Takeuchi et al. 2014). A significant increase in ED1 macrophages was observed in the heat group, occur- ring earlier than in the non-heat group. Moreover, there was a significant increase of Pax-7 (paired box protein 7) posi- tive satellite cells at 4 and 6 days after the crush injury in the heat group (Fig. 2a). Hence, the authors suggested that acceleration of the degeneration process (faster increase in ED1 macrophages) by heating could be a solution to pro- mote regeneration. Similar results were reported in another study, where heating stimulated satellite cell proliferation and protein synthesis during the regeneration of injured skeletal muscle in rats (Kojima et al. 2007). A recent study examined the effects of heat intervention (20 min, 40 °C, after injury) on muscle fiber regeneration after muscle injury (i.e., injection of cardiotoxin) in rats (El-Sheikh et al. 2022). In the heating group, the regenerating myotubes (defined as those with two or more central nuclei) were observed at three days post-injury, whereas in the non-heating group, they were observed at seven days post-injury. Hence, heat treatment hastens skeletal muscle regeneration. Moreover, passive muscle heating (hot packs, 42 °C, 20 min) acceler- ates the increase in myoblast determination protein (MyoD) and myogenin protein expression during the regeneration process in rats (Hatade et al. 2014). These proteins play piv- otal roles in myogenesis during embryonic/neonatal stages (Weintraub 1993) and muscle regeneration (Marsh et al. 1997). Additionally, applying heating shortly after muscle injury can accelerate the proliferation and differentiation of myogenic cells, promoting faster muscle repair (Hatade et al. 2014). It is well established that immobilization leads to a sig- nificant loss of muscle mass, strength and function in both elderly patients and injured athletes (Tipton 2015; Rom- mersbach et al. 2020). Furthermore, it has been shown that, in mice, heat exposures (40 °C, 30 min/day, 7 days) limit mitochondrial loss in denervated muscle, thus reducing the subsequent atrophy (Tamura et al. 2015). The interest in the impact of heat treatment on skeletal muscle regeneration in humans is increasing. A recent study examined the effects of 2-h daily heating (short-wave pulsed diathermy) during a 10-day immobilization period in humans. Heat intervention maintained mitochondrial function, attenuated atrophy, and increased the expression of PGC-1alpha (Hafen et al. 2019) (Fig. 2d). Furthermore, a significant increase in HSP70 and 90 protein content was found after immobilization in

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292 European Journal of Applied Physiology (2025) 125:277–301 the group using heat intervention only (Hafen et al. 2019) (Fig. 2b). HSPs play a chaperoning role: they prevent the loss of function of other proteins by assisting their correct folding/refolding and preventing protein denaturation and aggregation (Dahiya and Buchner 2019) (Fig. 2b). They can be activated by high temperature (heat stress), hypoxia (Gar- buz 2017) and exercise (Archer et al. 2018). Studies in rats indicate that HSPs exhibit important antioxidant properties and contribute to preserving muscle mass during immobili- zation periods (Selsby and Dodd 2005; Selsby et al. 2007). Among the HSP family, HSP70 has been shown to play a major role in the integrity of the muscle cell and its regen- eration after a traumatic event in mice (Senf 2013). With a deficiency of HSP70, there is an increase in inflamma- tion (macrophage infiltration in muscle fibers) and necrosis (Senf et al. 2013). Precisely, after injury (i.e., injection of cardiotoxin), average fiber CSA was lower and the num- ber of regenerating fibers containing centralized nuclei was higher in HSP70-deficient mice. Logically, a decreased mus- cle regeneration was also observed. Finally, a recent study examined the effects of whole-body heat intervention during two weeks of immobilization of a single lower leg (Labidi et al. 2024). The authors found that heat treatment prevented the decrease in maximal isometric strength of the plantar flexors and the decrease in the soleus CSA. Additionally, the FOXOs and NFκB (nuclear factor-kappa B) signaling pathways, which are involved in skeletal muscle atrophy, showed reduced activation with heat treatment. Thus, heat exposure appears as a promising intervention for mitigating the negative effects of immobilization in humans. Further- more, DNA methylation patterns are also altered by disuse atrophy. Fisher and coworkers showed that, by paralyzing the tibialis anterior muscle of rats, the promoter methylation of genes related to atrophy (i.e., muscle ring-finger protein-1 (MuRF1) and MAFbx/Atrogin-1) decreases (Fisher et al. 2017). Therefore, it is crucial to investigate the effects of single and repeated exercise and thermal interventions on the epigenome after immobilization. In summary, in animal models, cold intervention attenu- ates the oxi-inflammatory response following skeletal mus- cle injury, potentially leading to dysfunctional autophagy, which generates an incomplete degradation of organelles in the muscle cell in the early recovery phase. This mechanism might partly explain the observed delay in skeletal muscle regeneration with cold application. Conversely, heat inter- vention appears to be a promising treatment for treating muscle injury, promote regeneration, and preserve muscle mass and function. Considering the crucial role of muscles in overall physical function, these benefits could potentially translate into improved quality of life for individuals with cardiovascular disease (Monroe et al. 2020) or sarcopenic populations (Sanchez and Solsona 2021). Enhanced muscle health and function can lead to increased mobility, reduced fatigue, and improved ability to perform daily activities. This, in turn, can contribute to a higher level of independ- ence and overall well-being. Furthermore, the positive impact of heat treatment on muscle health may indirectly contribute to increased longevity. As cardiovascular disease and age-related muscle loss are associated with higher mor- tality rates, interventions that can mitigate these conditions may extend life expectancy. However, most of the studies are performed in non-human models (i.e., rat and mouse mod- els) and translation between species remains challenging. In this sense, more research in humans is needed to establish the optimal protocol for incorporating heat intervention into therapeutic approaches. The studies included in this section and treating about the effects of thermal interventions on skeletal muscle regeneration are summarized in Tables 3 (cold intervention) and 4 (heat intervention). Figure 2 details the effects of heat intervention on skeletal muscle regenera- tion during atrophy. Conclusions In conclusion, recent literature highlighted that thermal interventions can interfere with cellular adaptations involved in gains of physical aptitudes. Recent research showed that repeated post-resistance-exercise cold interventions inhibit several gains in hypertrophy, isometric and endurance strength. From a molecular viewpoint, the regular use of cryotherapy reduces oxi-inflammatory responses, down- regulates the MTORC1 pathway, ribosome biogenesis and regeneration. On the other hand, heat intervention can stimu- late protein synthesis, however, to date, no supplementary effect has been observed on skeletal muscle mass in trained humans. However, increases in isometric strength, blood volume and endurance performance have been observed in sub-elite and elite athletes. Importantly, heat intervention promotes muscle regeneration in the context of atrophy. In humans, heat treatment increases the expression of HSPs and PGC-1alpha, and decreases the autophagy pathway, which leads to preserved mitochondrial function and attenuated atrophy during immobilization. Therefore, heat intervention may represent an important tool for reducing atrophy and maintaining mitochondrial function during aging or immo- bilization. Furthermore, significant progress has been made in understanding the mechanisms underlying the effects of introducing heat treatment in the context of exercise and rehabilitation. In this way, epigenetic modifications such as DNA methylation and histone modifications might play a key role in the physiological adaptations induced by exercise and thermal interventions. Importantly, as blood markers represent an indirect measure, future studies must prioritize muscle tissue to evaluate the effects of thermal interventions. Further research should focus on determining the optimal

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293 European Journal of Applied Physiology (2025) 125:277–301 Table 3 Effects of cold intervention on skeletal muscle regeneration EIMD eccentric contraction-induced muscle damage, MCP1 monocyte chemoattractant protein 1, Pax7 paired box protein 7, TGF-β transforming growth factor beta, TNF-α tumor necrosis fac- tor α Study and design Main effects Injury or immobilization model Population and samples Recovery method Effects Takagi et al. 2011 Delayed macrophage migration Crush injury (30 s) Rats Ice pack – Lower increase in CSA and high proportion of centrally nucleated fibers Extensor Digitorum Longus 20 min; 0.3 ~ 1.3 °C Vieira Ramos et al. 2016 Decreased inflammatory processes (mac- rophage percentage and mRNA levels of inflammatory markers) Freezing injury (2 × 10 s) Rats (three-month-old males) Ice pack + No alteration of regeneration process (injury area, desmin and myoD expression) Tibialis anterior 3 × 30 min; immediately, 24 h and 48 h after injury; muscle surface temperature decreased by 17 °C Shibaguchi et al. 2016 Increase the development of fibrosis Bupivacaine injection Rats (8-week-old males) Ice pack – Delay in the timing of disappearance of TGF-β Soleus 20 min; 0 °C Singh et al. 2017 Delayed neutrophil and macrophage infiltra- tion Contusion injury Rats (12-week-old males) Ice pack ∅/– Higher percentage of immature myofibers Biceps Femoris 20 min; 0.3 ~ 1.3 °C No differences in CSA and capillary density Miyakawa et al. 2020 Inhibition of the accumulation of mac- rophages Crush injury (30 s) Rats (8-week-old males) Ice pack – Fewer neutrophils and MCP-1 + cells Extensor Digitorum Longus 20 min; 0.3 ~ 1.3 °C Kawashima et al. 2021 Persistence of necrotic muscle debris EIMD Mice (13–14 weeks old) Ice pack – Delayed macrophage M1 infiltration Gastrocnemius Delay in the emergence of Pax7 + myogenic cells 3 × 30 min; immediately, 24 h and 48 h after injury; muscle surface temperature decreased by 17 °C Miyazaki et al. 2023 Decrease in the accumulation of M1 mac- rophages Crush injury Rats Icing – Decrease in TNF-α expression Decrease in the number of myogenic precur- sor and the size of centrally nucleated regenerating myofibers Nagata et al. 2023 Decreased the accumulation of inducible nitric oxide synthase-expressing mac- rophages and the percentage of necrotic cells Milder crush injury (30 s) Rats (8-week-old males) Ice pack + Increased the number of activated satellite cells and cross-sectional area Extensor digitorum longus 3 × 30 min; immediately, 24 h and 48 h after injury; muscle surface temperature decreased by 16 °C

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294 European Journal of Applied Physiology (2025) 125:277–301 Table 4 Effects of heat intervention on skeletal muscle regeneration Study and design Main effects Injury or immobilization model Population and samples Recovery method Effects Goto et al. 2004 Stimulation of protein synthesis Hindlimb suspension (5 days) Rats (10-week-old males) Incubator + Increase in HSP72 expression Soleus 60 min; 41 °C Selsby and Dodd 2005 Increase in HSP25 and HSP72 expres- sion Bilateral immobilization in the plantar- flexed position (8 days) Rats (males) Heat chamber + Reduction in oxidative damage Soleus 30 min; every 2 days; 8 days; core tem- perature maintained at 41–41.5 °C Lower decrease in muscle mass Kojima et al. 2007 Stimulation of satellite cell proliferation and protein synthesis Cardiotoxin injection Rats (7-week-old males) Heat chamber + Tibialis anterior 60 min; 24 h before or immediately after injury; 41 °C Selsby et al. 2007 Potentiated muscle regrowth and reduced oxidant damage Bilateral immobilization in the plantar- flexed position (7 days) Male Rats Heat chamber + Increase of HSP25, HSP32, and HSP72 protein content Soleus 30 min; every 2 days; 7 days; core tem- perature maintained at 41–41.5 °C Touchberry et al. 2012 Increase in HSP72 expression and total protein concentration EIMD (Downhill running) Rats (10-week-old males) HWI + Decrease in CK plasma levels Soleus 20 min; 48 h prior to exercise; 43 °C Hatade et al. 2014 Faster increase of MyoD and myogenin protein expression Crush injury (30 s) Rats (8-week-old males) Hot pack with water + Extensor digitorum longus 20 min; 5 min after injury; 42 °C Takeuchi et al. 2014 Increase of ED1 macrophages earlier Crush injury (30 s) Rats (8-week-old males) Hot pack with water + Increase of Pax-7 positive satellite cells at 4 and 6 days after the crush injury Extensor digitorum longus 20 min; 5 min after injury; 42 °C Increase in CSA Nonaka et al. 2015 Increase in HSP25 and HSP72 expres- sion Atrophy induced by diabetes (injection of streptozotocin) Rats (12-week-old males) HWI + Lower decrease in CSA Extensor Digitorum Longus 30 min; 7 days after injection of strepto- zotocin and 5 days/week for 3 weeks; 42 °C Tamura et al. 2015 Limit mitochondrial and muscle mass loss Sciatic nerve transection Mice (8-week-old males) Heat chamber + Gastrocnemius 30 min/day for 1 week; 40 °C Shibaguchi et al. 2016 Decrease in fibrosis Bupivacaine injection Rats (8-week-old males) HWI + Increase in muscle mass, protein content and muscle fiber size Soleus 30 min; 48 h after injury and everyday for 12 days; 42 °C Hafen et al. 2019 Maintain mitochondrial function and increase the expression of PGC-1alpha Immobilization of the left leg (10 days) Healthy volunteers Short-wave pulsed diathermy + Increase in HSP70 and HSP90 Vastus lateralis 120 min everyday for 10 days; intra- muscular temperature increased by 4.2 ± 0.29 °C Lower decrease in CSA

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295 European Journal of Applied Physiology (2025) 125:277–301 CK creatine kinase, CD68 cluster of differenciation 68, KLF Krüppel-like family of transcription factors, HWI hot water bath, FoxO forkhead box O, MAFBX muscle atrophy F-box, mTOR mechanistic target of rapamycin, MyoD myoblast determination protein, NFκB nuclear factor-kappa B Table 4 (continued) Study and design Main effects Injury or immobilization model Population and samples Recovery method Effects Hirunsai and Srikuea 2020 Reduction of necrotic fibers, M1 mac- rophage invasion and TNFα protein expression Tenotomy (Achilles tendon ablation) Rats (10-week-old males) Thermal blankets + Increase of M2 macropage accumulation Soleus and plantaris 24 h before tenotomy and 3 days/week for 2 weeks; core temperature main- tained at 40.5–41.5 °C Hirunsai and Srikuea 2021 Lower decrease in CSA Tenotomy (Achilles tendon transection) Rats (10-week-old males) Thermal blankets + Decrease in autophagy-lysosomal sign- aling pathway Soleus and plantaris 30 min/day for 7 days; core temperature maintained at 40.5–41.5 °C AlSabagh et al. 2022 Lower decrase in CSA Atrophy induced by diabetes Rats (3-month-old males) Heat chamber + Decrease in muscle atrophy markers (CD68, KLF, MAFbx) 30 min/day; everyday for 6 weeks; 42 °C, 15.0 ± 2.0% RH Increase in muscle hypertrophy markers (AKT, mTOR, HSP70) El-Sheikh et al. 2022 Earlier increase of regenerating myo- tubes Cardiotoxin injection Rats How water bottle + Tibialis anterior 20 min; 5 min after injury; 40 °C Labidi et al. 2024 Lower decrease in maximal isometric strength and CSA Single-lower leg immobilization (2 weeks) Male participants Whole-body heat therapy + Lower activation of FoxO and NFκB signaling pathways Soleus and gastrocnemius

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296 European Journal of Applied Physiology (2025) 125:277–301 timing and the optimal “stress” of these interventions to pro- mote gains in performance and to develop complementary therapeutic approaches. This research topic is essential to enhance skeletal muscle adaptations and to mitigate muscle wasting in various pathological states and during immobi- lization periods. Acknowledgements The authors thank Dr. Glaviole for his advice. Author contributions Conceived and designed the study: TNG, RS, HB, VD, SR, FB, and AMJS. Analyzed the data of the literature: TNG, RS, HB, VD, SR, FB, and AMJS. Designed the tables and figures: TNG, RS, and AMJS. Wrote the paper: TNG, RS, and AMJS. All the authors read the manuscript and approved the final version. Funding Open access funding provided by University of Lausanne. No funding was received for the preparation of this work. Declarations Conflict of interest The authors confirm that this article’s content has no financial or non-financial interests that are directly or indirectly re- lated to the work submitted for publication. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Aboodarda SJ, George J, Mokhtar AH, Thompson M (2011) Muscle strength and damage following two modes of variable resistance training. J Sports Sci Med 10:635–642 Aguiar PF, Magalhães SM, Fonseca IAT et al (2016) Post-exercise cold water immersion does not alter high intensity interval training-induced exercise performance and Hsp72 responses, but enhances mitochondrial markers. Cell Stress Chaperones 21:793–804. https://doi.org/10.1007/s12192-016-0704-6 AlSabagh AT, Rao MS, Renno WM (2022) The impact of heat therapy on neuromuscular function and muscle atrophy in diabetic rats. Front Physiol 13:1039588. https://doi.org/10.3389/fphys.2022. 1039588 Archer AE, Von Schulze AT, Geiger PC (2018) Exercise, heat shock proteins and insulin resistance. Philos Trans R Soc B Biol Sci 373:20160529. https://doi.org/10.1098/rstb.2016.0529 Argus CK, Broatch JR, Petersen AC et al (2017) Cold-water immer- sion and contrast water therapy: no improvement of short-term recovery after resistance training. Int J Sports Physiol Perform 12:886–892. https://doi.org/10.1123/ijspp.2016-0127 Bagley JR, Burghardt KJ, McManus R et al (2020) Epigenetic responses to acute resistance exercise in trained vs. sedentary men. J Strength Cond Res 34:1574–1580. https://doi.org/10. 1519/JSC.0000000000003185 Barrès R, Yan J, Egan B et al (2012) Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab 15:405–411. https://doi.org/10.1016/j.cmet.2012.01.001 Bartke T, Schneider R (2020) You are what you eat—how nutrition and metabolism shape the genome through epigenetics. Mol Metab 38:100987. https://doi.org/10.1016/j.molmet.2020.100987 Bartoli M, Richard I (2005) Calpains in muscle wasting. Int J Biochem Cell Biol 37:2115–2133. https://doi.org/10.1016/j.biocel.2004. 12.012 Bartolomé I, Siquier-Coll J, Pérez-Quintero M et al (2021) 3-Week passive acclimation to extreme environmental heat (100± 3 °C) in dry sauna increases physical and physiological performance among young semi-professional football players. J Therm Biol 100:103048. https://doi.org/10.1016/j.jtherbio.2021.103048 Bleakley CM, Glasgow P, Webb MJ (2012) Cooling an acute muscle injury: can basic scientific theory translate into the clinical setting? Br J Sports Med 46:296–298. https://doi.org/10.1136/ bjsm.2011.086116 Botella J, Shaw CS, Bishop DJ (2023) Autophagy and exercise: cur- rent insights and future research directions. Int J Sports Med. https://doi.org/10.1055/a-2153-9258 Broatch JR, Petersen A, Bishop DJ (2014) Postexercise cold water immersion benefits are not greater than the placebo effect. Med Sci Sports Exerc 46:2139–2147. https://doi.org/10.1249/MSS. 0000000000000348 Broatch JR, Petersen A, Bishop DJ (2018) The influence of post- exercise cold-water immersion on adaptive responses to exer- cise: a review of the literature. Sports Med 48:1369–1387. https://doi.org/10.1007/s40279-018-0910-8 Broatch JR, Petersen A, Bishop DJ (2017) Cold-water immersion fol- lowing sprint interval training does not alter endurance signal- ing pathways or training adaptations in human skeletal muscle. Am J Physiol Regul Integr Comp Physiol 313:R372–R384. https://doi.org/10.1152/ajpregu.00434.2016 Brownstein AJ, Ganesan S, Summers CM, et al (2017) Heat stress causes dysfunctional autophagy in oxidative skeletal muscle. Physiol Rep 5:e13317. https://doi.org/10.14814/phy2.13317 Carnio S, LoVerso F, Baraibar MA et al (2014) Autophagy impair- ment in muscle induces neuromuscular junction degeneration and precocious aging. Cell Rep 8:1509–1521. https://doi.org/ 10.1016/j.celrep.2014.07.061 Castets P, Lin S, Rion N et al (2013) Sustained activation of mTORC1 in skeletal muscle inhibits constitutive and starva- tion-induced autophagy and causes a severe, late-onset myo- pathy. Cell Metab 17:731–744. https://doi.org/10.1016/j.cmet. 2013.03.015 Chaillou T, Treigyte V, Mosely S et al (2022) Functional impact of post-exercise cooling and heating on recovery and training adaptations: application to resistance, endurance, and sprint exercise. Sports Med Open 8:37. https://doi.org/10.1186/ s40798-022-00428-9 Cheng AJ, Willis SJ, Zinner C et al (2017) Post-exercise recovery of contractile function and endurance in humans and mice is accelerated by heating and slowed by cooling skeletal muscle. J Physiol 595:7413–7426. https://doi.org/10.1113/JP274870 Cheung K, Hume P, Maxwell L (2003) Delayed onset muscle sore- ness: treatment strategies and performance factors. Sports Med 33:145–164. https://doi.org/10.2165/00007256-20033 3020-00005 Choo HC, Lee M, Yeo V et al (2022) The effect of cold water immersion on the recovery of physical performance revisited:

Page 21

297 European Journal of Applied Physiology (2025) 125:277–301 a systematic review with meta-analysis. J Sports Sci 40:2608– 2638. https://doi.org/10.1080/02640414.2023.2178872 Cortés-Mancera FM, Sarno F, Goubert D, Rots MG (2022) Gene- targeted DNA methylation: towards long-lasting reprogram- ming of gene expression? In: Jeltsch A, Jurkowska RZ (eds) DNA methyltransferases—role and function. Springer, Cham, pp 515–533 Cui J, Gao Z, Leuenberger UA et al (2022) (2022) Repeated warm water baths decrease sympathetic activity in humans. J Appl Physiol 133:234–245. https://doi.org/10.1152/japplphysiol. 00684.2021 Dahiya V, Buchner J (2019) Chapter one—functional principles and regulation of molecular chaperones. In: Donev R (ed) Advances in protein chemistry and structural biology. Academic Press, New York, pp 1–60 de Mendoza A, Nguyen TV, Ford E et al (2022) Large-scale manipula- tion of promoter DNA methylation reveals context-specific tran- scriptional responses and stability. Genome Biol 23:163. https:// doi.org/10.1186/s13059-022-02728-5 Delatte B, Fuks F (2013) TET proteins: on the frenetic hunt for new cytosine modifications. Brief Funct Genom 12:191–204. https:// doi.org/10.1093/bfgp/elt010 Denham J, Marques FZ, Bruns EL et al (2016) Epigenetic changes in leukocytes after 8 weeks of resistance exercise training. Eur J Appl Physiol 116:1245–1253. https://doi.org/10.1007/ s00421-016-3382-2 Dimond JL, Roberts SB (2016) Germline DNA methylation in reef corals: patterns and potential roles in response to environmental change. Mol Ecol 25:1895–1904. https://doi.org/10.1111/mec. 13414 Earp JE, Hatfield DL, Sherman A et al (2019) Cold-water immer- sion blunts and delays increases in circulating testosterone and cytokines post-resistance exercise. Eur J Appl Physiol 119:1901– 1907. https://doi.org/10.1007/s00421-019-04178-7 Ehlert T, Simon P, Moser DA (2013) Epigenetics in Sports. Sports Med 43:93–110. https://doi.org/10.1007/s40279-012-0012-y El-Sheikh AA, El-Kordy EA, Issa SA (2022) Influence of heat treat- ment on muscle recovery after skeletal muscle injury in rats: histological and immunohistochemical studies. J Microsc Ultra- struct 10:63–71. https://doi.org/10.4103/jmau.jmau_85_20 Figueiredo VC, Roberts LA, Markworth JF, et al (2016) Impact of resistance exercise on ribosome biogenesis is acutely regulated by post-exercise recovery strategies. Physiol Rep. https://doi.org/ 10.14814/phy2.12670 Fiorenza M, Hostrup M, Gunnarsson TP et al (2019) Neuromuscular fatigue and metabolism during high-intensity intermittent exer- cise. Med Sci Sports Exerc 51:1642–1652. https://doi.org/10. 1249/MSS.0000000000001959 Fisher AG, Seaborne RA, Hughes TM et al (2017) Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle. FASEB J 31:5268–5282. https://doi.org/10. 1096/fj.201700089RR Frery P, Ruffault A, Esculier J-F et al (2023) L’utilisation du froid intense par les patients en traumatologie du sport: étude par ques- tionnaire en ligne auprès de 696 sportifs. J De Traumatologie Du Sport 40:21–26. https://doi.org/10.1016/j.jts.2023.01.006 Fuchs CJ, Kouw IWK, Churchward-Venne TA et al (2020) Postexercise cooling impairs muscle protein synthesis rates in recreational athletes. J Physiol 598:755–772. https://doi.org/10.1113/JP278 996 Fyfe JJ, Broatch JR, Trewin AJ et al (2019) Cold water immersion attenuates anabolic signaling and skeletal muscle fiber hyper- trophy, but not strength gain, following whole-body resistance training. J Appl Physiol 127:1403–1418. https://doi.org/10.1152/ japplphysiol.00127.2019 Garbuz DG (2017) Regulation of heat shock gene expression in response to stress. Mol Biol 51:352–367. https://doi.org/10.1134/ S0026893317020108 Goto K, Honda M, Kobayashi T et al (2004) Heat stress facilitates the recovery of atrophied soleus muscle in rat. Jpn J Physiol 54:285–293. https://doi.org/10.2170/jjphysiol.54.285 Goto K, Oda H, Kondo H et al (2011) Responses of muscle mass, strength and gene transcripts to long-term heat stress in healthy human subjects. Eur J Appl Physiol 111:17–27. https://doi.org/ 10.1007/s00421-010-1617-1 Hafen PS, Abbott K, Bowden J et al (2019) Daily heat treatment main- tains mitochondrial function and attenuates atrophy in human skeletal muscle subjected to immobilization. J Appl Physiol 127:47–57. https://doi.org/10.1152/japplphysiol.01098.2018 Hafen PS, Preece CN, Sorensen JR et al (2018) Repeated exposure to heat stress induces mitochondrial adaptation in human skeletal muscle. J Appl Physiol 125:1447–1455. https://doi.org/10.1152/ japplphysiol.00383.2018 Hao Y, Cui Y, Gu X (2016) Genome-wide DNA methylation profiles changes associated with constant heat stress in pigs as meas- ured by bisulfite sequencing. Sci Rep 6:27507. https://doi.org/ 10.1038/srep27507 Haq A, Ribbans WJ, Hohenauer E, Baross AW (2022) The effect of repetitive whole body cryotherapy treatment on adaptations to a strength and endurance training programme in physically active males. Front Sports Act Living 4:834386. https://doi.org/10. 3389/fspor.2022.834386 Hatade T, Takeuchi K, Fujita N et al (2014) Effect of heat stress soon after muscle injury on the expression of MyoD and myogenin during regeneration process. J Musculoskelet Neuronal Interact 14:325–333 He X-J, Chen T, Zhu J-K (2011) Regulation and function of DNA methylation in plants and animals. Cell Res 21:442–465. https:// doi.org/10.1038/cr.2011.23 Héberlé É, Bardet AF (2019) Sensitivity of transcription factors to DNA methylation. Essays Biochem 63:727–741. https://doi.org/ 10.1042/EBC20190033 Henderson KN, Killen LG, O’Neal EK, Waldman HS (2021) The cardi- ometabolic health benefits of sauna exposure in individuals with high-stress occupations a mechanistic review. Int J Environ Res Public Health 18:1105. https://doi.org/10.3390/ijerph18031105 Hesketh K, Shepherd SO, Strauss JA et al (2019) Passive heat therapy in sedentary humans increases skeletal muscle capillarization and eNOS content but not mitochondrial density or GLUT4 content. Am J Physiol Heart Circ Physiol 317:H114–H123. https://doi. org/10.1152/ajpheart.00816.2018 Hirunsai M, Srikuea R (2021) Autophagy-lysosomal signaling responses to heat stress in tenotomy-induced rat skeletal muscle atrophy. Life Sci 275:119352. https://doi.org/10.1016/j.lfs.2021. 119352 Hirunsai M, Srikuea R (2020) Heat stress ameliorates tenotomy- induced inflammation in muscle-specific response via regulation of macrophage subtypes. J Appl Physiol 128:612–626. https:// doi.org/10.1152/japplphysiol.00594.2019 Hohenauer E, Taeymans J, Baeyens J-P et al (2015) The effect of post- exercise cryotherapy on recovery characteristics: a systematic review and meta-analysis. PLoS ONE 10:e0139028. https://doi. org/10.1371/journal.pone.0139028 Horgan BG, Halson SL, Drinkwater EJ et al (2023) No effect of repeated post-resistance exercise cold or hot water immersion on in-season body composition and performance responses in academy rugby players: a randomised controlled cross-over design. Eur J Appl Physiol 123:351–359. https://doi.org/10.1007/ s00421-022-05075-2

Page 22

298 European Journal of Applied Physiology (2025) 125:277–301 Huang L, Li M, Deng C et al (2022) Potential therapeutic strategies for skeletal muscle atrophy. Antioxidants (Basel) 12:44. https://doi. org/10.3390/antiox12010044 Hung T-C, Liao Y-H, Tsai Y-S et al (2018) Hot water bathing impairs training adaptation in elite teen archers. Chin J Physiol 61:118– 123. https://doi.org/10.4077/CJP.2018.BAG560 Ihsan M, Labidi M, Racinais S (2023) Skeletal muscle oxidative adap- tations following localized heat therapy. Eur J Appl Physiol 123:1629–1635. https://doi.org/10.1007/s00421-023-05159-7 Jessop P, Toledo-Rodriguez M (2018) Hippocampal TET1 and TET2 expression and DNA hydroxymethylation are affected by physi- cal exercise in aged mice. Front Cell Dev Biol 6:45. https://doi. org/10.3389/fcell.2018.00045 Jin C, Ye J, Yang J et al (2019) mTORC1 mediates lysine-induced satellite cell activation to promote skeletal muscle growth. Cells 8:1549. https://doi.org/10.3390/cells8121549 Kakigi R, Naito H, Ogura Y et al (2011) Heat stress enhances mTOR signaling after resistance exercise in human skeletal muscle. J Physiol Sci 61:131–140. https://doi.org/10.1007/ s12576-010-0130-y Kaluhiokalani JP, Wallace TE, Ahmadi M et al (2024) Six weeks of localized passive heat therapy elicits some exercise-like improvements in resistance artery function. J Physiol. https:// doi.org/10.1113/JP286567 Kangaspeska S, Stride B, Métivier R et al (2008) Transient cyclical methylation of promoter DNA. Nature 452:112–115. https:// doi.org/10.1038/nature06640 Kawashima M, Kawanishi N, Tominaga T et al (2021) Icing after eccentric contraction-induced muscle damage perturbs the dis- appearance of necrotic muscle fibers and phenotypic dynam- ics of macrophages in mice. J Appl Physiol 130:1410–1420. https://doi.org/10.1152/japplphysiol.01069.2020 Kellmann M, Bertollo M, Bosquet L et al (2018) Recovery and per- formance in sport: consensus statement. Int J Sports Physiol Perform 13:240–245. https://doi.org/10.1123/ijspp.2017-0759 Kenttä G, Hassmén P (1998) Overtraining and recovery. a conceptual model. Sports Med 26:1–16. https://doi.org/10.2165/00007 256-199826010-00001 Kim K, Reid BA, Casey CA et al (2020) Effects of repeated local heat therapy on skeletal muscle structure and function in humans. J Appl Physiol 128:483–492. https://doi.org/10.1152/ japplphysiol.00701.2019 Kojima A, Goto K, Morioka S et al (2007) Heat stress facilitates the regeneration of injured skeletal muscle in rats. J Orthop Sci 12:74–82. https://doi.org/10.1007/s00776-006-1083-0 Kozłowska M, Kortas J, Żychowska M et al (2021) Beneficial effects of whole-body cryotherapy on glucose homeostasis and amino acid profile are associated with a reduced myostatin serum concentration. Sci Rep 11:7097. https://doi.org/10.1038/ s41598-021-86430-9 Kuligowski LA, Lephart SM, Giannantonio FP, Blanc RO (1998) Effect of whirlpool therapy on the signs and symptoms of delayed-onset muscle soreness. J Athl Train 33:222–228 Labidi M, Ihsan M, Behan FP et al (2021) Six weeks of localized heat therapy does not affect muscle mass, strength and contrac- tile properties in healthy active humans. Eur J Appl Physiol 121:573–582. https://doi.org/10.1007/s00421-020-04545-9 Labidi M, Racinais S, Alhammoud M et al (2024) 501 EP041—heat therapy during immobilization and rehabilitation: a proactive approach to preventing muscle atrophy and strength loss upon return to sport. Br J Sports Med 58:A93–A94. https://doi.org/ 10.1136/bjsports-2024-IOC.164 Lane SC, Camera DM, Lassiter DG et al (2015) Effects of sleep- ing with reduced carbohydrate availability on acute training responses. J Appl Physiol 119:643–655. https://doi.org/10. 1152/japplphysiol.00857.2014 Li J, Huang Q, Sun M et al (2016) Global DNA methylation varia- tions after short-term heat shock treatment in cultured micro- spores of Brassica napus cv. Topas Sci Rep 6:38401. https:// doi.org/10.1038/srep38401 Lindholm ME, Marabita F, Gomez-Cabrero D et al (2014) An inte- grative analysis reveals coordinated reprogramming of the epi- genome and the transcriptome in human skeletal muscle after training. Epigenetics 9:1557–1569. https://doi.org/10.4161/ 15592294.2014.982445 Liu J, Feng L, Li J, He Z (2015) Genetic and epigenetic control of plant heat responses. Front Plant Sci 6:267. https://doi.org/10. 3389/fpls.2015.00267 Lopez P, Radaelli R, Taaffe DR et al (2021) Resistance training load effects on muscle hypertrophy and strength gain: system- atic review and network meta-analysis. Med Sci Sports Exerc 53:1206–1216. https://doi.org/10.1249/MSS.0000000000 002585 MacInnis MJ, Gibala MJ (2017) Physiological adaptations to interval training and the role of exercise intensity. J Physiol 595:2915– 2930. https://doi.org/10.1113/JP273196 Mackey AL, Kjaer M, Dandanell S et al (2007) The influence of anti- inflammatory medication on exercise-induced myogenic pre- cursor cell responses in humans. J Appl Physiol 103:425–431. https://doi.org/10.1152/japplphysiol.00157.2007 Malabarba J, Windels D, Xu W, Verdier J (2021) Regulation of DNA (de)methylation positively impacts seed germination during seed development under heat stress. Genes (Basel) 12:457. https://doi. org/10.3390/genes12030457 Malm C (2001) Exercise-induced muscle damage and inflammation: fact or fiction? Acta Physiol Scand 171:233–239. https://doi.org/ 10.1046/j.1365-201x.2001.00825.x Malta ES, Dutra YM, Broatch JR et al (2021) The effects of regu- lar cold-water immersion use on training-induced changes in strength and endurance performance: a systematic review with meta-analysis. Sports Med 51:161–174. https://doi.org/10.1007/ s40279-020-01362-0 Marchant ED, Kaluhiokalani JP, Wallace TE et al (2022) Localized heat therapy improves mitochondrial respiratory capacity but not fatty acid oxidation. Int J Mol Sci 23:8500. https://doi.org/10. 3390/ijms23158500 Marsh DR, Criswell DS, Carson JA (1997) Myogenic regulatory fac- tors during regeneration of skeletal muscle in young, adult, and old rats. J Appl Physiol 83:1270–1275. https://doi.org/10.1152/ jappl.1997.83.4.1270 McGorm H, Roberts LA, Coombes JS, Peake JM (2018) Turning up the heat: an evaluation of the evidence for heating to promote exercise recovery, muscle rehabilitation and adaptation. Sports Med 48:1311–1328. https://doi.org/10.1007/s40279-018-0876-6 Meeusen R, Lievens P (1986) The use of cryotherapy in sports injuries. Sports Med 3:398–414. https://doi.org/10.2165/00007256-19860 3060-00002 Méline T, Mathieu L, Borrani F et al (2019) Systems model and indi- vidual simulations of training strategies in elite short-track speed skaters. J Sports Sci 37:347–355. https://doi.org/10.1080/02640 414.2018.1504375 Méline T, Solsona R, Antonietti J-P et al (2021) Influence of post-exer- cise hot-water therapy on adaptations to training over 4 weeks in elite short-track speed skaters. J Exerc Sci Fit 19:134–142. https://doi.org/10.1016/j.jesf.2021.01.001 Méline T, Watier T, Sanchez AM (2017) Cold water immersion after exercise: recent data and perspectives on “kaumatherapy.” J Physiol 595:2783–2784. https://doi.org/10.1113/JP274169 Minett GM, Costello JT (2015) Specificity and context in post-exer- cise recovery: it is not a one-size-fits-all approach. Front Physiol 6:130. https://doi.org/10.3389/fphys.2015.00130

Page 23

299 European Journal of Applied Physiology (2025) 125:277–301 Mittal M, Siddiqui MR, Tran K et al (2014) Reactive oxygen spe- cies in inflammation and tissue injury. Antioxid Redox Signal 20:1126–1167. https://doi.org/10.1089/ars.2012.5149 Miyakawa M, Kawashima M, Haba D et al (2020) Inhibition of the migration of MCP-1 positive cells by icing applied soon after crush injury to rat skeletal muscle. Acta Histochem 122:151511. https://doi.org/10.1016/j.acthis.2020.151511 Miyazaki A, Kawashima M, Nagata I et al (2023) Icing after skel- etal muscle injury decreases M1 macrophage accumulation and TNF-α expression during the early phase of muscle regeneration in rats. Histochem Cell Biol 159:77–89. https://doi.org/10.1007/ s00418-022-02143-8 Møller AB, Vendelbo MH, Schjerling P et al (2019) Immobilization decreases FOXO3a phosphorylation and increases autophagy- related gene and protein expression in human skeletal muscle. Front Physiol 10:736. https://doi.org/10.3389/fphys.2019.00736 Monroe JC, Song Q, Emery MS et al (2020) Acute effects of leg heat therapy on walking performance and cardiovascular and inflam- matory responses to exercise in patients with peripheral artery disease. Physiol Rep 8:e14650. https://doi.org/10.14814/phy2. 14650 Moore E, Fuller JT, Buckley JD et al (2022) Impact of cold-water immersion compared with passive recovery following a single bout of strenuous exercise on athletic performance in physically active participants: a systematic review with meta-analysis and meta-regression. Sports Med 52:1667–1688. https://doi.org/10. 1007/s40279-022-01644-9 Morris JG, Nevill ME, Boobis LH et al (2005) Muscle metabolism, temperature, and function during prolonged, intermittent, high-intensity running in air temperatures of 33 degrees and 17 degrees C. Int J Sports Med 26:805–814. https://doi.org/10. 1055/s-2005-837448 Nagata I, Kawashima M, Miyazaki A et al (2023) Icing after skeletal muscle injury with necrosis in a small fraction of myofibers lim- its inducible nitric oxide synthase-expressing macrophage inva- sion and facilitates muscle regeneration. Am J Physiol Regul Integr Comp Physiol 324:R574–R588. https://doi.org/10.1152/ ajpregu.00258.2022 Newham DJ, McPhail G, Mills KR, Edwards RH (1983) Ultrastructural changes after concentric and eccentric contractions of human muscle. J Neurol Sci 61:109–122. https://doi.org/10.1016/0022- 510x(83)90058-8 Nonaka K, Une S, Akiyama J (2015) Heat stress attenuates skeletal muscle atrophy of extensor digitorum longus in streptozotocin- induced diabetic rats. Acta Physiol Hung 102:293–300. https:// doi.org/10.1556/036.102.2015.3.7 Normand-Gravier T, Britto F, Launay T et al (2022a) Exercise dose equalization in high-intensity interval training: a scoping review. Int J Environ Res Public Health 19:4980. https://doi.org/10.3390/ ijerph19094980 Normand-Gravier T, Solsona R, Sanchez AMJ (2022b) The key role of histone H3 trimethylation in skeletal muscle adaptation to exer- cise. J Physiol 600:4063–4065. https://doi.org/10.1113/JP283661 Pointon M, Duffield R, Cannon J, Marino FE (2011) Cold applica- tion for neuromuscular recovery following intense lower-body exercise. Eur J Appl Physiol 111:2977–2986. https://doi.org/10. 1007/s00421-011-1924-1 Pomponio L, Ertbjerg P (2012) The effect of temperature on the activ- ity of μ- and m-calpain and calpastatin during post-mortem stor- age of porcine longissimus muscle. Meat Sci 91:50–55. https:// doi.org/10.1016/j.meatsci.2011.12.005 Qaisar R, Bhaskaran S, Van Remmen H (2016) Muscle fiber type diver- sification during exercise and regeneration. Free Radic Biol Med 98:56–67. https://doi.org/10.1016/j.freeradbiomed.2016.03.025 Racinais S, Wilson MG, Périard JD (2017) Passive heat acclimation improves skeletal muscle contractility in humans. Am J Physiol Regul Integr Comp Physiol 312:R101–R107. https://doi.org/10. 1152/ajpregu.00431.2016 Reggiani C, Schiaffino S (2020) Muscle hypertrophy and muscle strength: dependent or independent variables? A provocative review. Eur J Transl Myol 30:9311. https://doi.org/10.4081/ ejtm.2020.9311 Roberts LA, Muthalib M, Stanley J et al (2015a) Effects of cold water immersion and active recovery on hemodynamics and recovery of muscle strength following resistance exercise. Am J Physiol Regul Integr Comp Physiol 309:R389-398. https://doi.org/10. 1152/ajpregu.00151.2015 Roberts LA, Raastad T, Markworth JF et al (2015b) Post-exercise cold water immersion attenuates acute anabolic signalling and long- term adaptations in muscle to strength training. J Physiol (Lond) 593:4285–4301. https://doi.org/10.1113/JP270570 Robinson MM, Dasari S, Konopka AR et al (2017) Enhanced pro- tein translation underlies improved metabolic and physical adaptations to different exercise training modes in young and old humans. Cell Metab 25:581–592. https://doi.org/10.1016/j. cmet.2017.02.009 Rodrigues P, Trajano GS, Wharton L, Minett GM (2020a) Mus- cle temperature kinetics and thermoregulatory responses to 42 °C hot-water immersion in healthy males and females. Eur J Appl Physiol 120:2611–2624. https://doi.org/10.1007/ s00421-020-04482-7 Rodrigues P, Trajano GS, Wharton L, Minett GM (2020b) Effects of passive heating intervention on muscle hypertrophy and neuro- muscular function: a preliminary systematic review with meta- analysis. J Therm Biol 93:102684. https://doi.org/10.1016/j. jtherbio.2020.102684 Romanello V, Sandri M (2021) The connection between the dynamic remodeling of the mitochondrial network and the regulation of muscle mass. Cell Mol Life Sci 78:1305–1328. https://doi.org/ 10.1007/s00018-020-03662-0 Rommersbach N, Wirth R, Lueg G et al (2020) The impact of dis- ease-related immobilization on thigh muscle mass and strength in older hospitalized patients. BMC Geriatr 20:500. https://doi. org/10.1186/s12877-020-01873-5 Sajjanar B, Siengdee P, Trakooljul N et al (2019) Cross-talk between energy metabolism and epigenetics during temperature stress response in C2C12 myoblasts. Int J Hyperth 36:775–783. https://doi.org/10.1080/02656736.2019.1639834 Sanchez AM, Candau R, Bernardi H (2019) Recent data on cellular component turnover: focus on adaptations to physical exercise. Cells 8:542. https://doi.org/10.3390/cells8060542 Sanchez AMJ, Bernardi H, Py G, Candau RB (2014) Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise. Am J Physiol Regul Integr Comp Physiol 307:R956-969. https://doi.org/10.1152/ajpregu.00187.2014 Sanchez AMJ, Candau R, Raibon A, Bernardi H (2015) Autophagy, a highly regulated intracellular system essential to skeletal muscle homeostasis—role in disease. Muscle Cell and Tissue, Exerc Alt Expo. https://doi.org/10.5772/60698 Sanchez AMJ, Candau RB, Csibi A et al (2012a) The role of AMP- activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis. Am J Physiol Cell Physiol 303:C475-485. https://doi.org/10.1152/ajpcell.00125.2012 Sanchez AMJ, Csibi A, Raibon A et al (2012b) AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1. J Cell Biochem 113:695– 710. https://doi.org/10.1002/jcb.23399 Sanchez AMJ, Solsona R (2021) Chapter 9—mitophagy in sarco- penic muscle and practical recommendations for exercise train- ing. In: Sakuma K (ed) Sarcopenia. Elsevier, Amsterdam, pp 207–229

Page 24

300 European Journal of Applied Physiology (2025) 125:277–301 Sandri M, Coletto L, Grumati P, Bonaldo P (2013) Misregulation of autophagy and protein degradation systems in myopathies and muscular dystrophies. J Cell Sci 126:5325–5333. https://doi.org/ 10.1242/jcs.114041 Sautillet B, Bourdillon N, Millet GP, et al (2023) Hot water immer- sion: maintaining core body temperature above 38.5 °C mitigates muscle fatigue. Scand J Med Sci Sports. https://doi.org/10.1111/ sms.14503 Schoenfeld BJ, Ogborn D, Krieger JW (2017) Dose-response relation- ship between weekly resistance training volume and increases in muscle mass: a systematic review and meta-analysis. J Sports Sci 35:1073–1082. https://doi.org/10.1080/02640414.2016.1210197 Scoon GSM, Hopkins WG, Mayhew S, Cotter JD (2007) Effect of post-exercise sauna bathing on the endurance performance of competitive male runners. J Sci Med Sport 10:259–262. https:// doi.org/10.1016/j.jsams.2006.06.009 Seaborne RA, Sharples AP (2020) The interplay between exercise metabolism, epigenetics, and skeletal muscle remodeling. Exerc Sport Sci Rev 48:188–200. https://doi.org/10.1249/JES.00000 00000000227 Seaborne RA, Strauss J, Cocks M et al (2018) Methylome of human skeletal muscle after acute and chronic resistance exercise train- ing, detraining and retraining. Sci Data 5:180213. https://doi.org/ 10.1038/sdata.2018.213 Selsby JT, Dodd SL (2005) Heat treatment reduces oxidative stress and protects muscle mass during immobilization. Am J Physiol Regul Integr Comp Physiol 289:R134–R139. https://doi.org/ 10.1152/ajpregu.00497.2004 Selsby JT, Rother S, Tsuda S et al (2007) Intermittent hyperthermia enhances skeletal muscle regrowth and attenuates oxidative damage following reloading. J Appl Physiol 102:1702–1707. https://doi.org/10.1152/japplphysiol.00722.2006 Senf SM (2013) Skeletal muscle heat shock protein 70: diverse func- tions and therapeutic potential for wasting disorders. Front Physiol 4:330. https://doi.org/10.3389/fphys.2013.00330 Senf SM, Howard TM, Ahn B et al (2013) Loss of the inducible Hsp70 delays the inflammatory response to skeletal muscle injury and severely impairs muscle regeneration. PLoS ONE 8:e62687. https://doi.org/10.1371/journal.pone.0062687 Shibaguchi T, Sugiura T, Fujitsu T et al (2016) Effects of icing or heat stress on the induction of fibrosis and/or regeneration of injured rat soleus muscle. J Physiol Sci 66:345–357. https:// doi.org/10.1007/s12576-015-0433-0 Singh DP, Barani Lonbani Z, Woodruff MA et al (2017) Effects of topical icing on inflammation, angiogenesis, revasculariza- tion, and myofiber regeneration in skeletal muscle following contusion injury. Front Physiol 8:93. https://doi.org/10.3389/ fphys.2017.00093 Sitkowski D, Cisoń T, Szygula Z et al (2022) Hematological adap- tations to post-exercise sauna bathing with no fluid intake: a randomized cross-over study. Res Q Exerc Sport 93:795–803. https://doi.org/10.1080/02701367.2021.1921684 Solsona R, Borrani F, Bernardi H, Sanchez AMJ (2022) Perspec- tives on epigenetic markers in adaptation to physical exercise. Microrna 11:91–94. https://doi.org/10.2174/221153661166622 0318140844 Solsona R, Méline T, Borrani F et al (2023) Active recovery vs hot- or cold-water immersion for repeated sprint ability after a strenuous exercise training session in elite skaters. J Sports Sci. https://doi.org/10.1080/02640414.2023.2259267 Solsona R, Normand-Gravier T, Borrani F et al (2024) DNA meth- ylation changes during a sprint interval exercise performed under normobaric hypoxia or with blood flow restriction: a pilot study in men. Physiol Rep 12:e16044. https://doi.org/10. 14814/phy2.16044 Solsona R, Pavlin L, Bernardi H, Sanchez AM (2021) Molecular regulation of skeletal muscle growth and organelle biosynthe- sis: practical recommendations for exercise training. Int J Mol Sci 22:2741. https://doi.org/10.3390/ijms22052741 Solsona R, Sanchez AMJ (2020) Ribosome biogenesis and resistance training volume in human skeletal muscle. J Physiol (Lond) 598:1121–1122. https://doi.org/10.1113/JP279490 Solsona R, Sanchez AMJ (2021) Exercise and ribosome biogenesis in skeletal muscle hypertrophy: impact of genetic and epige- netic factors. J Physiol 599:3803–3805. https://doi.org/10. 1113/JP281984 Symons IK, Bruce L, Main LC (2023) Impact of overtraining on cognitive function in endurance athletes: a systematic review. Sports Med Open 9:69. https://doi.org/10.1186/ s40798-023-00614-3 Szustakowski JD, Kosinski PA, Marrese CA et al (2007) Dynamic resolution of functionally related gene sets in response to acute heat stress. BMC Mol Biol 8:46. https://doi.org/10.1186/ 1471-2199-8-46 Takagi R, Fujita N, Arakawa T et al (2011) Influence of icing on mus- cle regeneration after crush injury to skeletal muscles in rats. J Appl Physiol 110:382–388. https://doi.org/10.1152/japplphysi ol.01187.2010 Takeuchi K, Hatade T, Wakamiya S et al (2014) Heat stress pro- motes skeletal muscle regeneration after crush injury in rats. Acta Histochem 116:327–334. https://doi.org/10.1016/j.acthis. 2013.08.010 Tamura Y, Kitaoka Y, Matsunaga Y et al (2015) Daily heat stress treatment rescues denervation-activated mitochondrial clear- ance and atrophy in skeletal muscle. J Physiol 593:2707–2720. https://doi.org/10.1113/JP270093 Tamura Y, Matsunaga Y, Masuda H et al (2014) Postexercise whole body heat stress additively enhances endurance training- induced mitochondrial adaptations in mouse skeletal mus- cle. Am J Physiol Regul Integr Comp Physiol 307:R931-943. https://doi.org/10.1152/ajpregu.00525.2013 Tetievsky A, Assayag M, Ben-Hamo R et al (2014) Heat acclimation memory: do the kinetics of the deacclimated transcriptome predispose to rapid reacclimation and cytoprotection? J Appl Physiol 117:1262–1277. https://doi.org/10.1152/japplphysiol. 00422.2014 Tipton KD (2015) Nutritional support for exercise-induced injuries. Sports Med 45(Suppl 1):S93-104. https://doi.org/10.1007/ s40279-015-0398-4 Touchberry CD, Gupte AA, Bomhoff GL et al (2012) Acute heat stress prior to downhill running may enhance skeletal muscle remodeling. Cell Stress Chaperones 17:693–705. https://doi. org/10.1007/s12192-012-0343-5 Trybulski R, Stanula A, Vovkanych A, et al (2024) Optimal duration of cold and heat compression for forearm muscle biomechanics in mixed martial arts athletes: a comparative study. Med Sci Monit 30:e944149-1–e944149-11. https://doi.org/10.12659/ MSM.944149 Tsuchida W, Iwata M, Akimoto T et al (2017) Heat stress modulates both anabolic and catabolic signaling pathways preventing dexamethasone-induced muscle atrophy in vitro. J Cell Physiol 232:650–664. https://doi.org/10.1002/jcp.25609 Vaile J, Halson S, Gill N, Dawson B (2008) Effect of hydrotherapy on the signs and symptoms of delayed onset muscle soreness. Eur J Appl Physiol 102:447–455. https://doi.org/10.1007/ s00421-007-0605-6 Versey NG, Halson SL, Dawson BT (2013) Water immersion recov- ery for athletes: effect on exercise performance and practical recommendations. Sports Med 43:1101–1130. https://doi.org/ 10.1007/s40279-013-0063-8

Page 25

301 European Journal of Applied Physiology (2025) 125:277–301 Vieira Ramos G, Pinheiro CM, Messa SP et al (2016) Cryotherapy reduces inflammatory response without altering muscle regen- eration process and extracellular matrix remodeling of rat mus- cle. Sci Rep 6:18525. https://doi.org/10.1038/srep18525 Vizoso M, van Rheenen J (2021) Diverse transcriptional regulation and functional effects revealed by CRISPR/Cas9-directed epi- genetic editing. Oncotarget 12:1651–1662. https://doi.org/10. 18632/oncotarget.28037 Wackerhage H, Schoenfeld BJ, Hamilton DL et al (2019) Stimuli and sensors that initiate skeletal muscle hypertrophy following resistance exercise. J Appl Physiol 126:30–43. https://doi.org/ 10.1152/japplphysiol.00685.2018 Wall BT, Dirks ML, van Loon LJC (2013) Skeletal muscle atrophy during short-term disuse: implications for age-related sarco- penia. Ageing Res Rev 12:898–906. https://doi.org/10.1016/j. arr.2013.07.003 Walters BK, Read CR, Estes AR (2018) The effects of resistance training, overtraining, and early specialization on youth athlete injury and development. J Sports Med Phys Fitness 58:1339– 1348. https://doi.org/10.23736/S0022-4707.17.07409-6 Watanabe K, Koch Esteves N, Gibson OR et al (2024) Heat-related changes in the velocity and kinetic energy of flowing blood influence the human heart’s output during hyperthermia. J Physiol 602:2227–2251. https://doi.org/10.1113/JP285760 Weintraub H (1993) The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75:1241–1244. https://doi.org/10. 1016/0092-8674(93)90610-3 Weyrich A, Yasar S, Lenz D, Fickel J (2020) Tissue-specific epige- netic inheritance after paternal heat exposure in male wild guinea pigs. Mamm Genome 31:157–169. https://doi.org/10.1007/ s00335-020-09832-6 Widmann M, Nieß AM, Munz B (2019) Physical exercise and epige- netic modifications in skeletal muscle. Sports Med 49:509–523. https://doi.org/10.1007/s40279-019-01070-4 Wilson JM, Loenneke JP, Jo E et al (2012) The effects of endurance, strength, and power training on muscle fiber type shifting. J Strength Cond Res 26:1724–1729. https://doi.org/10.1519/JSC. 0b013e318234eb6f Yamane M, Teruya H, Nakano M et al (2006) Post-exercise leg and forearm flexor muscle cooling in humans attenuates endurance and resistance training effects on muscle performance and on circulatory adaptation. Eur J Appl Physiol 96:572–580. https:// doi.org/10.1007/s00421-005-0095-3 Zembron-Lacny A, Morawin B, Wawrzyniak-Gramacka E et al (2020) Multiple cryotherapy attenuates oxi-inflammatory response fol- lowing skeletal muscle injury. Int J Environ Res Public Health 17:E7855. https://doi.org/10.3390/ijerph17217855 Zhang Y, Chen Y, Xu Z et al (2020) Chronic exercise mediates epige- netic suppression of L-type Ca2+ channel and BKCa channel in mesenteric arteries of hypertensive rats. J Hypertens 38:1763– 1776. https://doi.org/10.1097/HJH.0000000000002457 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Effects of thermal interventions on skeletal muscle adaptations and regeneration: perspectives on epigenetics: a narrative review

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