Implications of Heat Stress-induced Metabolic Alterations for Endurance Training

Keywords

carbohydrate - metabolism - endurance training - heat acclimation - lipid - endurance athlete

Introduction

Relative to exercise completed in temperate conditions (18–21°C), environmental heat stress (>30°C) increases core temperature [1], heart rate [1] [2], circulating catecholamines [3], peripheral blood flow, sweat rate, and dehydration [4]. Concomitant to cardiovascular and thermoregulatory alterations, substrate metabolism shifts toward increased carbohydrate utilization and decreased lipid oxidation [5]. This increased physiological strain induced by heat stress during endurance exercise impairs exercise capacity and performance [6], warranting strategies to offset the impact of heat stress, including; heat acclimation/acclimatization protocols and specific nutritional considerations to ensure substrate availability, hydration and performance [7].

Increasingly, international sporting events (e. g. Tokyo Olympic Games and Doha World Athletics Championships) are hosted in cities with ambient temperatures sufficiently high to impair endurance performance (>12–13°C for Marathon Running) [8]. To counteract the effects of elevated ambient temperatures, athletes undergo “heat stress camps” to induce a heat-acclimated phenotype, leading to improved exercise capacity in hot conditions [9]. Heat acclimation aside, endurance training in elevated ambient temperatures may augment physiological stress and optimize metabolic adaptations [10]. This hypothesis is, in part, based on the notion that post-exercise adaptations are induced via metabolic demand, substrate depletion and potentially direct heat stress during endurance training, all amplified by environmental heat stress. This review will provide a brief overview of the metabolic impact of heat stress during endurance exercise and, against this backdrop, discuss the impact of heat-induced metabolic alterations on post-exercise molecular adaptation.

Effects of acute heat stress on substrate metabolism

Extensive research exists regarding the effect of heat stress on substrate metabolism during prolonged exercise ([Table 1), and it] has been reviewed elsewhere [5]. During submaximal exercise performed in elevated ambient temperature, the respiratory exchange ratio (RER) typically increases compared to thermoneutral conditions [3] [11] [12] [13] [14] [15], indicating a shift toward increased carbohydrate oxidation and decreased lipid oxidation. Reported increases in carbohydrate utilization during heat stress and exercise implemented fixed external workloads within a narrow range of relative exercise intensities between environmental conditions, typically 50–75% of maximal aerobic capacity (VO_2max) [11] [16] [17] [18] [19] [20] [21] [22], power (W_max) [23] [24] [25] and speed (V_max) [26]. Furthermore, when exercise intensity or ambient temperature is too low, several studies failed to alter substrate utilization in response to heat stress [21] [27] [28] [29].

Table 1 Overview of the methodological details and outcomes of studies investigating the effect of heat stress on prolonged exercise substrate metabolism.

Reference	Participants	Protocol	Metabolic effect (compared to temperate condition)	Thermoregulatory effects (compared to temperate)
Bennett et al. [41]	24 endurance trained males (˙VO2max, 62.3±6.6 mL·kg-1·min-1), 2 hr post absorptive following controlled pre-trial meal.	30-min capacity test consisting of 6×4 min 50 s steady state interspersed with 10 s sprints.	↑ Blood Glucose↑ Blood Lactate↑ Blood Alanine	↑ T rec (~ 0.5°C)
Charoensap et al. [34]	10 endurance trained males (˙VO2max, 58.1±6.8 mL·kg-1·min-1, first ventilatory threshold [VT1], 204±46 W), overnight fast	90-min cycling at 95% of HR associated with VT1 in 18°C and 33°C	↔︎ Mean HR↓ Energy Expenditure↓ CHO Oxidation ↔  Fat Oxidation↔︎ Adrenaline↔︎ Noradrenaline↔︎ HSP70	↔︎ T rec ↑ Estimated T mus (~0.7°C)
Dolny and Lemon, [16]	8 healthy males, (˙VO2max, 55±8 mL·kg-1·min-1), 4 hr post absorptive	90-min cycle at 65% ˙VO2max in 5, 20, 30°C	↑ RER ↑ CHO oxidation (~ 6.4%)↑ Blood Lactate↑ Serum adrenaline: noradrenaline↔︎ NEFA	↑ T rec (~ 0.25°C)
Febbraio et al. [14]	7 endurance trained males, (˙VO2max, 65±13 mL·kg-1·min-1), overnight fast	40-min cycle at 70% ˙VO2max in 20 & 40°C	↑ RER ↓ ˙VO2 ↑ CHO oxidation (~16.3%) ↑ net muscle glycogenolysis↑ Blood and Muscle Lactate↑ Blood Glucose↑ Plasma adrenaline	↑ T rec (~ 1°C)↑ T mus (~ 1.3°C)
Febbraio et al. [11]	12 endurance trained males, (˙VO2max, 65±7 mL·kg-1·min-1), overnight fast	40-min cycle at 70% ˙VO2max in 20 & 40°C	↑ RER ↔︎ ˙VO2 ↑ Net muscle glycogenolysis ↑ Net muscle CrP degradation ↑ Net muscle Cr accumulation ↑ Blood and Muscle Lactate↑ Blood Glucose ↔︎ Muscular ATP, ADP, AMP, IMP↑ Muscular NH3	↑ T rec (~ 1°C)↑ T mus (~ 1.7°C)
Fernandez-Elias et al. [18]	7 endurance trained males, (˙VO2max, 55±3 mL·kg-1·min-1), controlled pre-trial diet	Dehydrating exercise in heat, 4-hr rehydration, 40-min cycle at 75% ˙VO2max in 25, 36°C	↑ CHO oxidation ↑ Net muscle glycogenolysis ↑ Blood Lactate	↑ T rec (~ 0.7°C)
Fink, Costill & Van Handel[39]	6 physically active men	3×15-min cycling at 70 to 85% VO2max interspersed by 10 min (biopsies were collected) at 9 vs 41°C	↑ Net muscle glycogenolysis ↓ IMTG Utilization ↑ Blood Lactate↑ Blood Glucose↓ Blood Triglycerides	↑ T rec (~ 2°C)
Galloway and Maughan [19]	8 active males, (˙VO2max, ~56±7 mL·kg-1·min-1), overnight fast	Cycle TTE at 75% ˙VO2max in 4, 11, 21, 31°C	↔︎ CHO Oxidation↔︎ Blood Lactate↔︎ Blood Glucose↔︎ Glycerol↔︎ NEFA	↑ T rec (~ 0.3–0.5°C)
Hargreaves et al. [18]	6 endurance trained males, (˙˙VO2max, ~64 mL·kg-1·min-1), overnight fast	40-min cycle at 65% ˙VO2max in 20 & 40°C	↑ RER ↑ CHO Oxidation (~19.8%)↔︎ ˙VO2 ↑ Muscle glycogenolysis oxidation (~16.8%)↑ Hepatic Glucose Production ↑ Plasma Glucose & Lactate↑ Plasma Adrenaline: noradrenaline ↑ Plasma Cortisol↑ Plasma Glucagon↑ Plasma GH	↑ T rec (~ 0.9°C)
Hettinga et al. [23]	6 well-trained males, (˙VO2max, ~66 mL·kg-1·min-1), 2 hr fast	20-min cycle at 60% MAP in 15.5 & 35.5°C	↔︎ RER↑ ˙VO2↓ Gross Efficiency↑ Blood Lactate	↑ T rec (~ 0.3°C)
Jentjens et al. [38]	9 endurance trained males, (˙VO2max, 65±3 mL·kg-1·min-1), overnight fast	90-min cycle at 55% Wmax in 16 & 35°C with ~1.5 g·min-1 CHO ingestion	(Data from between 60–90 min of exercise)↔︎ RER↔︎ ˙VO2↔︎ Total CHO Oxidation↑ Exogenous CHO Oxidation↔︎ Fat Oxidation↑ Muscle Glycogenolysis↑ Plasma Lactate↔︎ Plasma Glucose↔︎ Insulin↔︎ NEFA	↑ T rec (~ 0.8°C)
Marino et al. [26]	9 endurance trained males, (˙VO2max, 66±4 mL·kg-1·min-1), nutritional status unknown	30-min run at 70% PTRS in 15 & 35°C	↑ RER ↔︎ ˙VO2↑ CHO Oxidation ↔︎ Plasma Lactate	↑ T rec (~0.6°C)
Maunder et al. [35]	Part A: 9 endurance trained males (˙VO2max, 57±5 mL·kg-1·min-1), overnight fast Part B: 11 Endurance Trained Males (˙VO2max, 57±5 mL·kg-1·min-1), 4 hr fast	Part A: 60-min cycling at absolute power output at VT1 in 18 & 35°C. Part B: 20-min cycling at absolute power output at VT1 in 18, 28, 34 & 40°C	Part A:↑ CHO Oxidation at high intensity only↓ Fat Oxidation at high intensity only↑ Plasma Lactate↔︎ Plasma GlucosePart B:↑ Plasma Adrenaline↑ CHO Oxidation at high and moderate intensity↓ Fat Oxidation at high intensity	Part A:↑ T rec (data not reported)↑ T mus (data not reported)Part B:↑ T rec (~ 0.2 and 0.3°C at moderate and high intensity respectively)↑ T mus
Nielsen et al. [29]	7 healthy males, (˙VO2max, ~54 mL·kg-1·min-1), overnight fast	30-min incline walk in 18°C followed immediately by 60-min incline walk in 40°C	↑ ˙VO2↔︎ Arterial & Venous Lactate↔︎ Plasma Glucose↔︎ Plasma FFA↔︎ Muscle Glycogenolysis	↑ T es (~1.1°C)↑ T sk (~4.8°C)
Parkin et al. [13]	8 endurance trained males, (˙VO2max, 55±8 mL·kg-1·min-1), overnight fast	Cycle TTE at 70% ˙VO2max in 3, 20, 40°C	↔︎ RER ↔︎ ˙VO2↑ Net Muscle Glycogenolysis↑ Plasma Adrenaline↔︎ Noradrenaline	↑ T rec (~0.5°C)
Yaspelkis et al. [22]	9 endurances trained, heat acclimatized males, (˙VO2max, 69±1 mL·kg-1·min-1), overnight fast	60-min cycle at ~74% ˙VO2max in 24 & 34°C	↔︎ RER ↔︎ ˙VO2↔︎ CHO Oxidation↔︎ Net muscle glycogenolysis↑ Plasma Glucose &Lactate↔︎ Glycerol↔︎ NEFA	↑ T rec (~0.4°C)
Young et al. [15]	13 untrained males, (˙VO2max, 45±5 mL·kg-1·min-1), 4 hr post liquid meal	30-min cycle at 70% ˙VO2max in 21 & 49°C	↑ RER↓ ˙VO2↑ CHO Oxidation (~3.8%)↔︎ Net muscle glycogenolysis↑ Plasma and Muscle Lactate	↑ T rec (~0.7°C)

Esophageal temperature (T_es), Muscle temperature (T_mus), Rectal temperature (T_rec), Skin temperature (T_sk).

While there is a foundational understanding of the role of heat stress on exercise metabolism, matched external workload designs lack ecological validity and translational applicability as endurance athletes typically reduce their absolute workload when training in hot conditions [30] [31] [32] [33] [34]. Furthermore, these approaches do not permit the identification of exercise intensity or temperature (either core or ambient) thresholds likely to impact metabolism. Recently, Maunder et al. [35] investigated the relationship between exercise intensity, environmental heat stress, and carbohydrate oxidation during endurance cycling exercise. At lower exercise intensities (~68% VO_2max), higher ambient temperatures (40°C: 2.64±0.77 g·min-1) were required to stimulate carbohydrate oxidation rates compared to temperate conditions (18°C: 2.25±0.65 g·min^-1). Conversely, exercise at a higher relative intensity (~81% VO_2max) led to increases (+10.7%) in carbohydrate oxidation at lower environmental temperature (34°C: 3.74±0.74 g·min^-1) compared to temperate conditions (18°C: 3.38±0.40 g·min^-1). These findings underscore the limited insight into the impact of heat stress on substrate utilization during exercise using matched workload designs. Additionally, this highlights the need to adjust carbohydrate intake during endurance events in hot conditions, with particular attention to events or stages of a grand tour characterized by high-intensity periods (time trial and mountain stages of a grand tour) relative to cooler days or flatter stages of the race [36].

When exercise intensity was heart rate-matched (95% of HR associated with VT₁) between environmental conditions (18 vs. 33°C), Charoensap et al. [34] reported reduced power output (−17%) in hot conditions and a subsequent reduction in total energy expenditure during exercise compared to temperate conditions (-14%). Notably, fat oxidation rates were similar between environmental conditions, with a reduction in carbohydrate oxidation accounting for reduced energy expenditure in hot conditions. Moreover, a reduction in absolute power output attenuated any rise in core temperature, a critical factor in stimulating carbohydrate oxidation during heat-stressed exercise. Critically, following heat acclimation, athletes increase power output for a given heart rate under heat stress [37] [38], which requires greater rates of ATP synthesis, and by extension, carbohydrate oxidation, meaning the differences observed within the present study may not be consistent following a period of acclimation. While data from Maunder et al. [35] and Charoensap et al. [37] appear to represent a paradigm shift in understanding how substrate utilization is changed during exercise under heat stress, it is important to consider that while external workloads can be reduced during training or self-paced races (e. g. time trials), this is unlikely during race situations where intensity is dictated by other competitors.

The impact of heat stress during exercise on amino acid metabolism and protein turnover is yet to be directly investigated. However, indirect measures of protein breakdown, including urinary ammonia (NH₃) are elevated following heat-stressed exercise in both trained [11] and untrained individuals [39] supporting the notion that protein breakdown is increased during exercise in elevated ambient temperatures. Mechanistically, NH₃ is produced during deamination of adenosine 5′-monophosphate (AMP) to form NH₃ and inosine 5′-monophosphate (IMP) or from the oxidation of branched-chain amino acids (BCAA) within skeletal muscle [40]. Increased NH₃ accumulation without increased IMP indicates protein breakdown during exercise under heat stress [11], and while the relative contribution of liberated amino acids to energy production pathways during exercise is likely negligible, the impact on post-exercise recovery and skeletal muscle proteostasis is yet to be resolved. Recent work from our group characterized the metabolomic impact of maximal exercise under environmental heat stress using an ecologically valid self-paced exercise model [41]. Despite power output being significantly reduced during exercise in the heat, core temperature increased by ~1.5°C, resulting in significantly altered post-exercise serum metabolomes, including increased glycolytic metabolites (glucose, lactate and glucarate) and amino acids (alanine, glutamate and isoleucine). Increased circulating concentrations of alanine coupled with glucose and lactate are representative of the multi-tissue alanine cycle, whereby liberated alanine (from skeletal muscle) is transported via the bloodstream to the liver for gluconeogenesis [42]. Additionally, decreased glutamate and isoleucine may indicate the use of amino acids as tricarboxylic acid cycle precursors to sustain energy metabolism during high rates of glycolytic flux. It has been proposed that pyruvate dehydrogenase (PDH) flux is increased during exercise and heat stress [5] to support increased rates of carbohydrate oxidation. While this remains to be investigated, the alterations in glucogenic amino acids reported in our previous work highlight the often-overlooked role of amino acid metabolism in substrate provision and may indicate increased amino acid metabolism during exercise and heat stress ([Fig. 1]). The implementation of ecologically valid study designs, characterization of whole-body metabolism, and harnessing of -omics approaches, permitting a greater understanding of the molecular response to exercise and heat stress, are required to facilitate a step-change in sports nutrition guidelines. For instance, if amino acid metabolism or protein breakdown is increased during exercise in hot conditions, it may be appropriate to increase protein intake immediately following heat-stressed exercise. For further reading on the application and utility of omics in exercise physiology research, see [43] [44] [45].

Fig. 1 Overview of the alanine cycle encompassing liver and muscle metabolism. Alanine is liberated from proteins during proteolysis within skeletal muscle, which enters the bloodstream and is transported to the liver. Once in the liver, glutamate-pyruvate aminotransferase (ALT) converts alanine to pyruvate, which is then converted to glucose through gluconeogenesis, a process augmented under heat stress. Increased circulating alanine and urea may be hallmarks of protein breakdown during exercise and heat stress. Created with Biorender.com [rerif].

Regulation of substrate utilization during exercise under heat stress

Multiple proposed mechanisms exist to explain the shift in substrate utilization, characterized by an increase in carbohydrate oxidation in response to heat stress during exercise. These include increased circulating adrenaline [21] [35] [46], the direct effect of temperature on enzyme-controlled reactions (Q₁₀ Effect) [47], altered skeletal muscle fiber recruitment [27] [48], altered blood flow and reduced oxygen supply to muscles [49]. While each is a plausible mechanism and likely impacts substrate utilization, their relative contribution is difficult to quantify experimentally; nevertheless, each will be discussed here.

Hormonal regulation of substrate utilization – adrenaline

Circulating adrenaline is elevated during exercise [50] and is further augmented under heat stress [3] [12] [13] [20] [29] [51] [52] [53] [54]. Mechanistically, glycogen phosphorylase activity is increased by β-adrenergic receptor stimulation [55], leading to increased muscle glycogenolysis [3] [12] [51] [56]. One study reported no difference in muscle glycogenolysis, which is explained by experimental design [29]. Participants completed 30 minutes of exercise in cool conditions (18–20°C) before 60 minutes of exercise in hot conditions (40°C); as such, this study lacked sufficient counterbalancing between environmental conditions, rendering the resultant data difficult to interpret. Supporting the mechanistic role of adrenaline augmenting muscle glycogenolysis, trained men infused with adrenaline during exercise at 70% ˙VO_2max increased muscle glycogen utilization and lactate accumulation [57] despite no change in muscle nucleotide concentrations between environmental conditions (20 vs. 40°C) [11]. Moreover, increased RER during exercise in hot conditions is ubiquitous compared to thermoneutral conditions [3] [11] [16] [20] [27], consistent with metabolic responses observed during exercise with adrenaline infusion [57] [58].

While investigating the relationship between exercise intensity, heat stress, and substrate utilization, Maunder et al. [34] [35] provided supporting evidence of the role of adrenaline in inducing carbohydrate oxidation during heat-stressed exercise. Changes in carbohydrate oxidation tended to correlate with circulating adrenaline at moderate (r=0.35, P=0.07) and high (r=0.60, P=0.001) exercise intensities underpinned by changes in core and muscle temperatures [35]. Additionally, when rises in core temperature were blunted by reductions in absolute exercise intensity, a reduction in carbohydrate oxidation occurred, likely due to no changes in circulating adrenaline [34], as reported during work-matched studies [51].

Skeletal muscle aside, hyperglycemia is often observed during exercise in hot conditions [11] [14] [21] [41] [59], potentially due to adrenaline-induced hepatic glucose production (HGP). While limited evidence exists in humans, Howlett et al. reported increased circulating glucose and HGP when adrenaline is infused in physiological concentrations in endurance-trained [60] and bi-laterally adrenalectomized humans [61]. Despite requiring further investigation, this evidence highlights the potential regulatory role of adrenaline in blood glucose homeostasis and HGP during exercise and heat stress.

Counterintuitively, given the potent lipolytic regulation by adrenaline [62] [63] [64] during heat-stressed exercise, whole-body fat oxidation [34] and circulating plasma fatty acid concentrations were unchanged [21] [29] [52] [59] despite overall reductions in FFA uptake in working skeletal muscle [52] indicating reduced FFA release from adipocytes. This phenomenon may be explained by reduced adipose tissue blood flow when exercising in the heat, reducing available albumin for fatty acid transport, and promoting fatty acid re-esterification within adipocytes. However, similar plasma glycerol levels reported by Yaskpelkis et al. [21] during exercise and heat stress, may indicate that fatty acid esterification is unlikely to account for comparable FFA levels and is more likely due to reduced fatty acid lipolysis when exercising in the heat. Although not experimentally confirmed, the direct inhibitory effect of heat on hormone-sensitive lipase and other enzymes responsible for fatty acid liberation and metabolism should be considered.

Altered muscle fiber type recruitment

It has been reported that during exercise in hot conditions (49°C), individuals with a higher proportion of type II muscle fibers exhibited greater muscle lactate accumulation compared to those with greater type I density [27], spurring the hypothesis that type II fibers have greater sensitivity to heat stress, thus altering the metabolic impact of heat stress during exercise [27] [48]. To test this, Febbraio and colleagues utilized immunohistochemical analyses to characterize muscle glycogen use between fiber types following exercise and in hot conditions (40°C) [3]. Compared to exercise completed in temperate conditions (20°C), muscle lactate was greater following exercise in hot conditions. When considering fiber type-specific differences, muscle glycogen was significantly reduced in Type I muscle fibers after exercise in the heat, with no difference in Type II fibers. The fiber type-specific response observed was consistent with muscle glycogen utilization during prolonged exercise in temperate conditions [65] .

The direct effect of heat stress on skeletal muscle temperature (T_mus)

Exercise increases T _mus in a workload-dependent manner [66] [67] and is amplified with heat stress [3] [11] [20]. Theoretically, any rise in T _mus would directly alter enzyme activity and thus substrate metabolism [22] [47]. The temperature coefficient (Q₁₀) is the factor by which the rate of enzymatic reactions changes in response to increased/decreased temperature, meaning for every 10°C increase, a 2 to 3-fold increase in enzyme reaction rate is expected [68]. While a 10°C increase in T _mus is supra-physiological, a modest 2°C increase in T _mus may result in a 30 to 40% increase in enzyme activity.

Direct investigations of increased T _mus on intramuscular metabolism are scarce; however, Edwards et al. [69] demonstrated increased glycogen utilization and lactate accumulation following exhaustive isometric contractions following limb immersion in a water bath (44°C). While this study only intended to increase skeletal muscle temperature, it is noteworthy that core temperature was increased relative to temperate conditions. Given the stimulatory role of heat stress on circulating adrenaline concentrations the resultant increases in carbohydrate oxidation, may not be due to direct muscle temperature per se. To isolate the impact of muscle heating, Febbraio et al. used external heating pads (on the thigh) to elevate muscle temperature by 2°C immediately before supra-maximal exercise (2 min cycling at 115% ˙VO_2max) in active (but untrained) males [14]. Pre-exercise circulating adrenaline was not increased by muscle heating, but muscle glycogenolysis and lactate accumulation were increased post-exercise compared to exercise without pre-heating. Additionally, pre-heating increased the magnitude of ATP breakdown, with increased IMP and ammonia accumulation compared to non-heated muscle. Given these changes occurred in heated muscle only, the authors concluded that temperature per se increased carbohydrate utilization via anaerobic pathways. Mechanistically, heat-induced changes in total adenine nucleotide (TAN) pool (ATP, ADP, AMP) and IMP accumulation may result in altered carbohydrate metabolism via allosteric activation of phosphofructokinase (PFK) [70] and phosphorylase [71], two critical enzymes in glycogenolysis and glycolysis that increase glycolytic flux. In a follow-up study, the same research group completed a study where one leg was heated and the other cooled before and during exercise at 70% ˙VO_2max using water-perfused cuffs [72]. The initial difference in T _mus in the heated vs. cooled leg was reduced during exercise; however, it remained significantly elevated at the termination of exercise. While there was an increase in the glycogenolytic rate in the heated limb, no differences in high-energy phosphagen metabolism were noted between legs, providing evidence that T _mus per se has a role in regulating carbohydrate metabolism during exercise and heat stress.

Altered skeletal muscle blood flow

To facilitate thermoregulation during exercise and heat stress, blood flow is prioritized to working muscles, the skin and essential organs while splanchnic [49], hepatic [73], renal [74], and inactive muscle blood flow is reduced. Competing demands for blood supply mean that cardiovascular demand exceeds the maximal cardiac output capacity in the heat, leading to potentially altered substrate metabolism and ultimately impaired performance through reduced oxygen supply to the working muscle [22] [29] [47] [59]. It is contentious whether blood supply to working muscle is altered during exercise and heat stress [29] [75], and there is little understanding of how this alters local oxygen extraction. Direct measures of active limb blood flow via thermodilution plethysmography [29] [76] [77] [78] and doppler flowmetry [79] during exercise in the heat revealed unaltered skeletal blood flow. When implementing graded heat stress at rest and during exercise, Pearson et al. [80] reported gradual increases in leg blood flow (LBF), cardiac output, and leg vascular conductance (LVC), which correlated to muscle temperature at rest and during exercise (r ² =0.86–0.99; P<0.05). Muscle and skin perfusion were also increased, as evidenced by reductions in leg arteriovenous oxygen (a-vO₂) difference and increases in deep femoral venous O₂ content. Crucially, the authors used multiple approaches to validate whether heat stress increased subcutaneous and muscle vascular vasodilation.

More recently, near-infrared spectroscopy (NIRS) has revealed reduced muscle oxygenation during exercise under heat stress (40°C), with a simultaneous increase in skin blood flow providing strong evidence of a vascular shunt away from working muscles toward the skin for thermoregulation [81]. The authors also reported decreased muscle oxygen saturation and increased deoxygenated hemoglobin in heat stress conditions, indicating a widened arteriovenous VO₂ difference and a rise in oxygen extraction. A significant increase in deoxygenated hemoglobin may be interpreted as a limitation in oxygen delivery rather than an inability to utilize the available oxygen [82]. However, adjustments in deoxygenated hemoglobin indicate muscle oxygenation changes only when total hemoglobin volume is relatively stable, meaning caution should be used when interpreting the muscle oxygenation solely based on altered deoxygenated hemoglobin during the exercise. Discrepancies between the findings of these studies may be explained by the exercise protocols, whereby the latter was exhaustive cycling exercise compared to sub-maximal knee extensor exercise in the former.

When coupled with dehydration, muscle blood flow was attenuated during exercise [31]; however, the adverse effects of dehydration on cardiovascular function must be considered here, as function is severely impaired when compared to hyperthermia alone or exercise in temperate conditions [56]. When environmental heat stress (~35°C) was combined with dehydration during exercise, contracting limb blood flow decreased by ~1.0 L·min^-1, compared with comparable euhydrated exercise in the heat, with no difference in leg VO₂ [52]. Despite this, metabolic analysis highlighted an increase in muscle glycogen utilization and lactate accumulation with exercise and dehydration [52]. The data generated by this research group suggests that even if muscle blood flow is reduced during exercise and heat stress, arteriovenous oxygen difference is adjusted accordingly to ensure that oxygen supply is not compromised. While this evidence shows that oxygen supply is unlikely to play a significant role in altered muscle metabolism during exercise in the heat, it does not rule out the influence of decreased blood flow on underlying metabolic processes. The functional vascular shunt in skeletal muscle has already been shown to alter substrate metabolism due to an altered supply rate of nutrients and removal of metabolic by-products [83]. However, the importance of nutrient and non-nutrient supply and removal under heat stress has not been investigated directly.

Implications of Heat stress on the post-exercise Adaptive Response

During exercise, heat stress, a significant stressor transiently activates or inhibits signaling pathways that regulate cellular energy storage, metabolism, contraction, ion handling, and vascularization [10]. Several recent reviews have provided an overview of the benefits of heat therapy, including angiogenesis, muscle mass regulation, mitochondrial biogenesis, glucose metabolism and insulin signaling [84] and its potential usefulness to augment endurance training adaptations [85]. These reviews primarily focus on passive heating strategies before or after exercise. In contrast, the present review focuses on the combined effect of heat stress during exercise to optimize endurance training adaption.

Heat shock protein (HSP) response

Heat shock factors are activated in response to exercise, heat, inflammation and oxidative stress, promoting mRNA expression of their respective HSPs, a unique family of proteins responsible for ensuring multiple chaperone roles, ensuring correct protein folding and repairing damaged proteins [86]. Functionally, HSPs represent an evolutionary quality control mechanism allowing cells to ensure protein quality, and they are especially important during periods of cellular stress such as exercise or heat stress. In this context, the induction of HSP expression and subsequent increased capacity to offset the deleterious effects of heat stress on protein structure and function is referred to as thermotolerance. Notably, thermotolerance is increasingly viewed as a critical adaptive component of heat acclimation [87] [88] that is essential for allowing repeated heat exposures over a short timeframe.

After initial stimulus, HSP mRNA is transiently elevated, translating to robust increases in HSP protein expression following several weeks [89] [90]. Heat shock proteoforms with roles in exercise adaptation and cellular tolerance are HSP27, 60, 72, and 90. HSP72 has numerous chaperone roles, including transporting and folding newly synthesized polypeptides, which are folded structures within the process of protein synthesis and maintenance. Thus, HSP72 facilitates mitochondrial biogenesis and molecular exercise adaptation in response to heat stress and protein repair during recovery. Indeed, when exposed to 15 bouts of endurance training and passive heat stress, protein expression of HSP60 increased by 2.5 and 1.75-fold in mouse plantaris and soleus muscles, respectively, while 65- and 4-fold increases in HSP72 protein expression were reported in response to heat stress alone. When heat stress was preceded by exercise (30 min running at 25 m·min^-1), an additive effect of heat stress post-exercise was found [91]. Combining exercise and heat stress can induce greater HSP70 expression relative to either treatment alone [92], potentially improving thermotolerance in subsequent bouts of heat stress during exercise.

In humans, exercise-induced HSP responses appear to be intensity- and duration-dependent [93] [94] with no increase in HSP expression following 1 hour of exercise [34] and diminished magnitude and time-course of HSP response in individuals with higher basal HSP content [95] [96]. Following a period of heat acclimation, basal HSP72 protein expression increased by ~18% [97], with mRNA transiently increasing (+195%) following each bout of heat acclimation before returning to baseline within 24 hours [98] [99]. Increases in basal HSP expression may confer improved cellular thermotolerance, a hypothesis supported by impaired thermotolerance in murine HSP KO models [100].

Endurance-like adaptation and oxidative capacity

In C2C12 myotubes, heat stress increases AMP-activated protein kinase (AMPK) phosphorylation and mRNA expression of Sirtuin-1 (SIRT1) and PGC-1α [101] [102], a key regulator of mitochondrial biogenesis. Consequently, transcription of downstream transcription factors, such as nuclear respiratory factor (NRF) 1, NRF2, and mitochondrial transcription factor (Tfam), are upregulated, increasing mitochondrial components (Cycs, COXII, COXIV) and glucose transporter-4 (GLUT4) protein content. Repeated heat exposures (2 h at 40°C on 5 consecutive days) increased PGC-1α and mitochondrial subunit protein abundance compared to control cells (maintained at 37°C), and when exposed to a lipopolysaccharide (LPS) challenge, heat-acclimated cells maintained peak oxidation rates and exhibited greater oxidative capacity [102].

The application of heat stress in conjunction with endurance training has seldom been researched in humans and even less in highly trained subjects. Nevertheless, a single bout of aerobic cycling exercise (50% W_max) at 33°C in untrained individuals reduced PGC1-α mRNA expression compared to exercise at 20°C [103]. Additionally, three weeks of exercise (matched at a rating of perceived exertion of 15) in the heat did not increase PGC1-α mRNA expression compared to similar exercise in temperate conditions [104]. Crucially, in these studies, the impact of exercise intensity cannot be overlooked. In the former [104], power output was significantly reduced during exercise in hot conditions. In the case of the latter implementing walking exercise [105], the exercise intensity may have been insufficient to induce increases in PGC-1α or markers of mitochondrial biogenesis [106]. Interestingly, ˙VO_2peak increased in the group that trained at 20°C only, with no difference in peak power output between groups. The authors concluded that heat stress might limit the effectiveness of aerobic exercise to increase aerobic power and may blunt regular exercise-induced PGC1-α expression. The same group replicated its study in untrained women and reported improved aerobic power and PGC1-α expression following the heat acclimation protocol [107]. Given the blunted evaporative heat loss responses and augmented rise in core temperature in response to heat stress at a given absolute exercise intensity in women [108] [109], it may be appropriate to speculate that alterations in substrate metabolism and the intra-muscular adaptive response observed in males are exacerbated in females.

Ten days of active heat acclimation (i. e. walking at 30–40% ˙VO_2max twice for 45 min at 42°C, interspersed with 10 min rest) in recreationally active males (56.4±4.4 ml·kg·min^-1) and females (42.3±3.4 ml·kg·min^-1) increased HSP72 expression but did not enhance markers of mitochondrial biogenesis (CaMK, TFAM & PGC-1α protein expression) nor oxidative protein expression (COX I – IV) [105]. More recently, Maunder et al. [33] investigated the effects of 3 weeks of active heat acclimation (5 sessions per week ranging from moderate to severe exercise domains) on temperate exercise performance and metabolic adaptation in endurance trained males (53.4±7 ml·kg·min^-1). Compared to participants that exercised at 18°C, citrate synthase activity (a marker of mitochondrial biogenesis) increased by 1.25-fold following endurance training in the heat and was significantly correlated with training-induced change in time trial performance (r=0.51). Critically, training intensity was matched between environmental conditions based on relative cardiovascular demand at the first and second ventilatory thresholds, meaning physiological strain was maintained despite reductions in absolute workload in the heat.

Pro-angiogenic response to heat stress

The formation of new blood vessels from existing ones, called angiogenesis, is closely regulated by exercise [110] and is essential to optimize endurance performance [111]. Increasing the number of capillaries in skeletal muscle improves oxygen and nutrient exchange and enhances metabolic by-product removal leading to improved aerobic and anaerobic exercise capacity [112], ventilatory threshold, and critical power [113]. During exercise, elevated skeletal muscle blood flow increases shear stress within the muscle vasculature [114], triggering the release of pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet derived growth factor (PDGF) [115] [116]. Additionally, blood flow kinetics are altered when exercising in hot conditions. Heat increases local arteriolar vasodilation, increasing antegrade sheer stress and dilation of conduit arteries [117], which increases skeletal muscle VEGF release. Moreover, biochemical mechanisms induced by exercise and heat stress may also increase VEGF and other pro-angiogenic factors via increased reactive oxygen species (ROS) production [118] and increased production of lactate [119] [120].

It is widely accepted that heat stress increases endothelial nitric oxide synthase (eNOS) expression, which plays a crucial role in regulating vasomotor function and vascular remodeling in vitro [121] [122] [123] [124] and in rodents [125] [126] [127] [128] [129]. In humans, a single bout of heat stress increases endothelial mRNA expression of pro-angiogenic factors [116], while repeated exposures (6–8 weeks) to heat stress increase skeletal muscle eNOS content and induces angiogenesis [130] [131]. Interestingly, Hesketh et al. [131] reported that 6 weeks of passive heat stress (via heat chamber at 40°C) induced comparable angiogenesis to time-matched exercise alone (moderate intensity exercise, ~65% VO_2peak), albeit in untrained sedentary individuals. Additionally, recent work by Kaluhiokalani et al. [132] investigated the impact of 6 weeks of passive heat therapy (via short-wave diathermy) or exercise (knee extension exercise for 2 h, 3 days per week) on vascular function in previously untrained individuals. Blood flow during a passive leg movement increased to the same extent both in the exercise condition (~10.5%) and heat therapy condition (~8.5%). Peak vascular conductance was also increased in similar proportions (~25%) in both conditions. Meanwhile, exercise induced additional vascular adaptation with increased peak flow rate (~19%), capillary-to-fiber ratio, capillary density, and capillary-to-fiber perimeter exchange index compared to no change in heat therapy conditions.

When administered in isolation, passive heat therapy appears to provide some of the benefits of exercise spanning both mitochondrial and vascular adaptations. Nevertheless, it remains unclear whether combining heat stress and exercise would augment vascular adaptations further, and future studies should aim to understand the combined effects of exercise and heat stress on vascular adaptations.

Hematological adaptation to repeated heat stress

A well-documented adaptation to repeated heat stress, plasma volume expansion, is rapidly induced and occurs following relatively few exposures to heat stress (between 3 to 4 days) [133]. Ultimately, any increase in plasma volume has two physiological advantages; (1) increased vascular filling to support cardiovascular stability, and (2) increased specific heat of blood to lower skin blood flow responsiveness [134]. This increase in plasma volume precedes changes in hemoglobin concentration, thus leading to an initial relative decrease in hemoglobin and hematocrit [135]. Consequently, heat-induced erythropoiesis requires a more extended intervention period (>4 weeks) before red blood cell volume (RBCV) and hemoglobin mass (Hb_mass) are increased [136]. Increased RBCV and Hb_mass represent critical endurance training adaptations, improving oxygen transport, and contributing to improved VO_2max, a key determinant of endurance performance. Interestingly, when implementing prolonged heat-acclimation protocols (5 weeks) using either environmental chambers [137] [138] or ‘heat suits’ [139] in highly-trained participants (VO_2max ~ 75 ml·kg·min^-1), Hb_mass was increased compared to unheated controls. Crucially, this increase in Hb_mass improved peak power during incremental and time-trial cycling tests. While recent evidence supports the application of heat stress during exercise to promote hematological adaptation, several studies have failed to report increases in Hb_mass, albeit the heat acclimation protocols in these studies were considerably shorter [140] [141] [142], leaving limited time for erythropoiesis to compensate for hemodilution following plasma volume expansion. Crucially, longer heat exposures (weeks vs. days) are required for a marked increase in RBCV and Hb_mass. A summary of biomolecular adaptation in response to endurance exercise combined with heat exposure is proposed in [Fig. 2].

Fig. 2 Overview of heat-inducible molecular pathways associated with endurance training adaptation. In response to multiple cellular stressors, Heat shock factors (HSF) are activated and promote the transcription of heat shock proteins (HSP), which have numerous roles in promoting protein stability and functioning and as a quality control mechanism in cells. A key adaptive outcome of endurance training, mitochondrial biogenesis occurs in response to cellular and metabolic stress following substrate depletion and subsequent activation of AMPK, and the ‘master regulator’ of mitochondrial biogenesis PGC-1α. Augmented mitochondrial biogenesis has been reported in vitro and in vivo, albeit limited human evidence highlights the supplementary benefit of heat stress during exercise. Alterations in skeletal muscle blood flow associated with exercise and heat stress increase the expression of pro-angiogenic transcription factors. Whether heat stress promotes skeletal muscle microvascular adaptation in conjunction with exercise in trained individuals requires further investigation. Abbreviations: AMPK: AMP-activated protein kinase; eNOS: endothelial nitric oxide synthase; E3: Ubiquitin (Ub) Ligases; FOXO: Forkhead box O; HIF-1α: Hypoxia inducible factor-1α; HSF: Heat Shock Factor; HSP: Heat Shock Protein; PGC-1α: Peroxisome proliferator-activated receptor-gamma coactivator-1α; pO ₂ : partial pressure of oxygen; p53: tumor protein p53; ROS: reactive oxygen species; ULK1: Unc-51-like kinase 1; VEGF: vascular endothelial growth factor. Created with Biorender.com [rerif].

Future directions

It is well-documented that women are underrepresented in sports and exercise science research [143]. A meta-analysis of the physiological and performance adaptations to heat acclimation included 96 studies with 1,056 total participants, of which only 76 (7%) were female [144]. By comparison, a recent meta-analysis including studies only implementing heat acclimation in female included 22 articles with 235 participants [145]. Currently, the magnitude of whole-body adaptation to heat stress appears to be similar in both men and women, albeit the time course of specific adaptations and optimal strategies for exposure to heat stress remains unknown (for both sexes). The potential for gender-specific molecular adaptative responses also requires further investigation. For instance, in response to continuous and interval training, skeletal muscle HSP expression appears to be gender-specific, with 38 and 23% increases observed for men, respectively, and only 3 and 4% for women [146]. Notably, the diminished cryoprotective response observed in females may be attributable to the heat-protective effect of high estrogen levels [147], providing a mechanistic rationale for gender differences in HSP expression [148]. Only two studies have investigated the gender differences in HSP expression response to heat stress. Firstly, Gillum et al. reported increased HSP in males following a single bout of heat-stressed exercise compared to females during both the follicular and luteal phases [149]. When implementing a controlled hyperthermia acclimation protocol, there was no difference in HSP72 mRNA expression between males and females [150]. The contrasting findings can be attributed to different biological measures (mRNA vs. protein), hyperthermia induction (external vs. controlled hyperthermia), and exercise duration (acute vs. chronic).

Evidence of increased carbohydrate oxidation during combined heat stress and exercise has been studied in males only; further work is required to elucidate the impact of ambient temperature on substrate utilization in women. Additionally, given the blunted evaporative heat loss responses and augmented rise in core temperature in response to heat stress at a given absolute exercise intensity in women compared to men [108] [109], it may be appropriate to speculate that alterations in substrate metabolism observed in males are exacerbated in females. That said, all the studies in the present review have been conducted solely with males, and thus the impact of heat stress on exercise metabolism in females remains to be resolved.

Methodological developments, including applying mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy in exercise metabolism, provide an exciting opportunity for biochemical interrogation of the impact of heat stress during exercise beyond a targeted analysis of specific proteins or metabolites. Profiling biofluids (urine, sweat, serum and saliva) and tissue samples (muscle biopsies) provide high-resolution systemic and local insight into the impact of exercise and heat stress. For example, recent work has sought to characterize the serum exercise metabolome in response to exercise under environmental heat stress in trained participants [41]. Alterations to glycolytic metabolites were observed, but crucially, novel alterations to circulating amino acids (alanine and leucine) were identified between environmental conditions, highlighting potentially divergent protein requirements following exercise during heat stress.

Multiple mechanisms have been proposed for altered substrate utilization under heat stress, with both local (direct impact of heat stress on enzymatic reactions, fiber-type recruitment, and altered blood flow) and systemic (circulating adrenaline) factors considered responsible. Based on the current evidence, circulating adrenaline is critical in regulating substrate utilization during exercise under heat stress. Carbohydrate oxidation is increased when adrenaline is elevated during exercise [3] [12] [51] [56], and no alterations in substrate utilization are observed when rises in core temperature and, by extension, increased circulating adrenaline are blunted [34]. Further mechanistic insight is required to elucidate the relative contribution of these factors to substrate utilization. No in vitro evidence supports the hypothesis that adrenaline is a critical regulatory factor in skeletal muscle metabolism in response to heat stress. Future research should combine exercise mimetics such as electrical pulse stimulation [151] [152] [153] [154], heat stress, and adrenaline treatment in vitro to better understand each factor's relative contribution to altered substrate metabolism ([Fig. 3]).

Fig. 3 Overview of current opportunities and shortcomings within the current evidence base that, if addressed, would significantly advance the field of thermal exercise physiology and nutrition. Opportunities are shown in order of translational applicability from left to right. Research on women represents the highest priority regarding translational potential and need. Further to the right, future in vitro studies will provide critical insight into the mechanisms associated with heat stress and exercise, albeit with the least capacity for translation to practice. Created with Biorender.com [rerif].

Conclusion

Despite the increased prevalence of sporting competitions held in locations with high ambient temperatures, questions remain regarding the regulation of metabolism during exercise under heat stress and the subsequent impact on substrate metabolism. Alongside key modifiable training variables such as time, duration, frequency and modality, heat stress is a readily modifiable factor that alters substrate utilization and potentially benefits endurance training outcomes. The optimal strategy for inducing high muscle and core temperatures remains to be elucidated, and this lack of consensus makes providing practical advice regarding promoting endurance training adaptation difficult. Despite promising in vitro and pre-clinical data supporting the role of heat stress on endurance training adaptation, caution should always be used when extrapolating findings to humans. Resolving the role of heat stress on intra-muscular signaling responses and subsequent endurance training adaptation remains an emerging and exciting area of research and will continue to be topical as long as sporting events are held in hot environments and global temperatures continue to rise.

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