Equine metabolic investigation of the phosphodiesterase-4 inhibitor ibudilast as a potential performance enhancer.

RATIONALE: Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. METHODS: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. RESULTS: A total of 20 in vivo metabolites were identified under experimental conditions, including 12 Phase I and 8 Phase II conjugated metabolites. Phase I metabolites were predominantly formed through hydroxylation (mono-, di-, and tri-hydroxylation). Demethylated metabolites were also identified during this investigation. Additionally, the research detected Phase II metabolites conjugated with glucuronic and sulfonic acids. CONCLUSIONS: The data presented here can assist in detecting the PDE4 inhibitor ibudilast and uncover its illicit use in competitive sports.

Detection of FG-4592 and metabolites in equine plasma, urine and hair following oral administration.

FG-4592 is a hypoxia-inducible factor inhibitor that has been approved for therapeutic use in some countries. This class of compounds can increase the oxygen carrying capacity of the blood and thus have the potential to be used as performance enhancing agents in sports. The purpose of this study was to investigate the detection of FG-4592 and metabolites in equine plasma and mane hair following a multiple dose oral administration to two Thoroughbred racehorses, to identify the best analytical targets for doping control laboratories. Urine samples were also analysed, and the results compared to previously published urine data. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification in urine and plasma. Liquid chromatography-tandem mass spectrometry was used for full sample analysis of urine, plasma and hair samples and generation of urine and plasma profiles. FG-4592 and a mono-hydroxylated metabolite were detected in plasma. FG-4592 was detected with the greatest abundance and gave the longest duration of detection, up to 312 h post-administration, and would be the recommended target in routine doping samples. FG-4592 was detected in all mane hair samples collected post-administration, up to 166 days following the final dose, showing extended detection can be achieved with this matrix. To the best of the authors' knowledge, this is the first report of FG-4592 and metabolites in equine plasma and hair samples. Urine results were consistent with the previously published data, with FG-4592 offering the best target for detection and longest detection periods.

Investigations into the human metabolism of ecdysterone.

The possible performance-enhancing effects and medical benefits of ecdysterone (ECDY) have been discussed several times throughout the last decades. In 2020, the World Anti-Doping Agency include ECDY in their monitoring programme and continued this prevalence study until now. Only little is known about the human metabolism of ECDY besides the first study performed on human subjects in the field of sports drug testing that was already conducted in 2001. Aim of this study was the in-depth investigation on human ECDY metabolism to improve its detectability and to support the decision-making processes as to how ECDY can be implemented most effectively into sports drug testing regulations. In a first trial, one male volunteer was administered with threefold deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high-resolution/high-accuracy mass spectrometry. Samples were collected for up to 14 days, and metabolites excreted unconjugated, glucuronidated, and sulphated were investigated. The detected deuterated metabolites were confirmed in a second administration trial encompassing two male and one female volunteers. After the administration of 50 mg of unlabelled ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including side chain cleavage at C21. A large interindividual variation in the amounts of excreted metabolites was visible, and considerable differences in abundances of early- and late-excretion phase metabolites were observed.

The Acute Effects of a Single Dose of Molecular Hydrogen Supplements on Responses to Ergogenic Adjustments during High-Intensity Intermittent Exercise in Humans.

This research examined the effects of single-dose molecular hydrogen (H2) supplements on acid-base status and local muscle deoxygenation during rest, high-intensity intermittent training (HIIT) performance, and recovery. Ten healthy, trained subjects in a randomized, double-blind, crossover design received H2-rich calcium powder (HCP) (1500 mg, containing 2.544 µg of H2) or H2-depleted placebo (1500 mg) supplements 1 h pre-exercise. They performed six bouts of 7 s all-out pedaling (HIIT) at 7.5% of body weight separated by 40 s pedaling intervals, followed by a recovery period. Blood gases' pH, PCO2, and HCO3- concentrations were measured at rest. Muscle deoxygenation (deoxy[Hb + Mb]) and tissue O2 saturation (StO2) were determined via time-resolved near-infrared spectroscopy in the vastus lateralis (VL) and rectus femoris (RF) muscles from rest to recovery. At rest, the HCP group had significantly higher PCO2 and HCO3- concentrations and a slight tendency toward acidosis. During exercise, the first HIIT bout's peak power was significantly higher in HCP (839 ± 112 W) vs. Placebo (816 ± 108 W, p = 0.001), and HCP had a notable effect on significantly increased deoxy[Hb + Mb] concentration during HIIT exercise, despite no differences in heart rate response. The HCP group showed significantly greater O2 extraction in VL and microvascular (Hb) volume in RF during HIIT exercise. The HIIT exercise provided significantly improved blood flow and muscle reoxygenation rates in both the RF and VL during passive recovery compared to rest in all groups. The HCP supplement might exert ergogenic effects on high-intensity exercise and prove advantageous for improving anaerobic HIIT exercise performance.

Effort-based decision making in response to high-dose androgens: role of dopamine receptors.

INTRODUCTION: Anabolic-androgenic steroids (AAS) are performance-enhancing drugs used by both world-class and rank-and-file athletes. AAS abuse has been linked with risky decision-making, ranging from drunk driving to abusing multiple drugs. Our lab uses operant behavior in rats to test the effects of AAS (testosterone) on decision making. In our previous study, testosterone caused rats to work harder for food reward during an effort discounting (ED) task. ED is sensitive to dopamine in the nucleus accumbens, and AAS alter accumbens dopamine receptor expression. Accordingly, we determined if testosterone increases response to dopamine receptor antagonists during ED. METHODS: Rats were treated chronically with high-dose testosterone (7.5 mg/kg; n = 9) or vehicle (n = 9). We measured baseline preference for the large reward in an ED task, where rats choose between a small easy reward (one lever press for one sugar pellet) and a large difficult reward (2, 5, 10, or 15 presses for three pellets). Preference for the large reward was measured after administration of D1-like (SCH23390, 0.01 mg/kg) or D2-like (eticlopride, 0.06 mg/kg) receptor antagonists. RESULTS: At baseline, testosterone- and vehicle-treated rats showed similar preference for the large reward lever (FR5, testosterone: 68.6 ± 9.7% and vehicle: 85.7 ± 2.5%). SCH23390 reduced large reward preference significantly in both groups (FR5, testosterone: 41.3 ± 9.2%; vehicle: 49.1 ± 8.2%; F(1,16) = 17.7; P < 0.05). Eticlopride decreased large reward preference in both groups, but more strongly in testosterone-treated rats (FR5: testosterone: 37.0 ± 9.7%; vehicle: 56.3 ± 7.8%; F(1,16) = 35.3; P < 0.05). CONCLUSION: Testosterone increases response to dopamine D2-like receptor blockade, and this contributes to previously observed changes in decision-making behaviors.

In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples.

SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-9009 in an associated plasma and urine doping sample. Liquid chromatography-high-resolution mass spectrometry was used to verify the presence of the N-dealkylated metabolite (M1) in both matrices, with a low concentration of the parent compound and additional N-desalkyl metabolites (M2 and M3) detected in the plasma sample as supporting evidence of administration. To the best of the authors' knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.

Timing of Creatine Supplementation around Exercise: A Real Concern?

Creatine has been considered an effective ergogenic aid for several decades; it can help athletes engaged in a variety of sports and obtain performance gains. Creatine supplementation increases muscle creatine stores; several factors have been identified that may modify the intramuscular increase and subsequent performance benefits, including baseline muscle Cr content, type II muscle fibre content and size, habitual dietary intake of Cr, aging, and exercise. Timing of creatine supplementation in relation to exercise has recently been proposed as an important consideration to optimise muscle loading and performance gains, although current consensus is lacking regarding the ideal ingestion time. Research has shifted towards comparing creatine supplementation strategies pre-, during-, or post-exercise. Emerging evidence suggests greater benefits when creatine is consumed after exercise compared to pre-exercise, although methodological limitations currently preclude solid conclusions. Furthermore, physiological and mechanistic data are lacking, in regard to claims that the timing of creatine supplementation around exercise moderates gains in muscle creatine and exercise performance. This review discusses novel scientific evidence on the timing of creatine intake, the possible mechanisms that may be involved, and whether the timing of creatine supplementation around exercise is truly a real concern.

Mitochondria-targeted antioxidant supplementation improves 8 km time trial performance in middle-aged trained male cyclists.

BACKGROUND: Exercise increases skeletal muscle reactive oxygen species (ROS) production, which may contribute to the onset of muscular fatigue and impair athletic performance. Mitochondria-targeted antioxidants such as MitoQ, which contains a ubiquinone moiety and is targeted to mitochondria through the addition of a lipophilic triphenylphosphonium cation, are becoming popular amongst active individuals as they are designed to accumulate within mitochondria and may provide targeted protection against exercise-induced oxidative stress. However, the effect of MitoQ supplementation on cycling performance is currently unknown. Here, we investigate whether MitoQ supplementation can improve cycling performance measured as time to complete an 8 km time trial. METHOD: In a randomized, double-blind, placebo-controlled crossover study, 19 middle-aged (age: 44 ± 4 years) recreationally trained (VO2peak: 58.5 ± 6.2 ml·kg- 1·min- 1, distance cycled per week during 6 months prior to study enrollment: 158.3 ± 58.4 km) male cyclists completed 45 min cycling at 70% VO2peak followed by an 8 km time trial after 28 days of supplementation with MitoQ (20 mg·day- 1) and a placebo. Free F2-isoprostanes were measured in plasma samples collected at rest, after 45 min cycling at 70% VO2peak and after completion of the time trial. Respiratory gases and measures of rating of perceived exertion (RPE) were also collected. RESULTS: Mean completion time for the time trial was 1.3% faster with MitoQ (12.91 ± 0.94 min) compared to placebo (13.09 ± 0.95 min, p = 0.04, 95% CI [0.05, 2.64], d = 0.2). There was no difference in RPE during the time trial between conditions (p = 0.82) despite there being a 4.4% increase in average power output during the time trial following MitoQ supplementation compared to placebo (placebo; 270 ± 51 W, MitoQ; 280 ± 53 W, p = 0.04, 95% CI [0.49, 8.22], d = 0.2). Plasma F2-isoprostanes were lower on completion of the time trial following MitoQ supplementation (35.89 ± 13.6 pg·ml- 1) compared to placebo (44.7 ± 16.9 pg·ml- 1 p = 0.03). CONCLUSION: These data suggest that MitoQ supplementation may be an effective nutritional strategy to attenuate exercise-induced increases in oxidative damage to lipids and improve cycling performance.

Creatine Supplementation and Brain Health.

There is a robust and compelling body of evidence supporting the ergogenic and therapeutic role of creatine supplementation in muscle. Beyond these well-described effects and mechanisms, there is literature to suggest that creatine may also be beneficial to brain health (e.g., cognitive processing, brain function, and recovery from trauma). This is a growing field of research, and the purpose of this short review is to provide an update on the effects of creatine supplementation on brain health in humans. There is a potential for creatine supplementation to improve cognitive processing, especially in conditions characterized by brain creatine deficits, which could be induced by acute stressors (e.g., exercise, sleep deprivation) or chronic, pathologic conditions (e.g., creatine synthesis enzyme deficiencies, mild traumatic brain injury, aging, Alzheimer's disease, depression). Despite this, the optimal creatine protocol able to increase brain creatine levels is still to be determined. Similarly, supplementation studies concomitantly assessing brain creatine and cognitive function are needed. Collectively, data available are promising and future research in the area is warranted.

Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control.

The transcriptional activator hypoxia-inducible factor (HIF) is a vital arbitrator in the performance of cellular responses lacking oxygen supply in aerobic organisms. Because these compounds are capable of enhancing the organism's capacity for molecular oxygen transport, they possess great potential for abuse as a performance-enhancing agent in sports. A comprehensive study of the metabolic conversion of the most popular HIF stabilisers such as IOX2, IOX3 and IOX4 using equine liver microsomes (in vitro) is reported. The parents and their metabolites were identified and characterised by liquid chromatography-mass spectrometry in negative ionisation mode using a QExactive high-resolution mass spectrometer. Under the current experimental condition, a total of 10 metabolites for IOX2 (three phase I and seven phase II), nine metabolites for IOX3 (four phase I and five phase II) and five metabolites for IOX4 (three phase I and two phase II) were detected. The outcome of the present study is as follows: (1) all the three IOX candidates are prone to oxidation, results in subsequent monohydroxylated, and some dihydroxylated metabolites. (2) Besides oxidation, there is a possibility of hydrolysis and de-alkylation, which results in corresponding carboxylic acid and amide, respectively. (3) The glucuronide and sulphate conjugate of the parent drugs as well as the monohydroxylated analogues were observed in this study. The characterised in vitro metabolites can potentially serve as target analytes for doping control analysis.

The effect of ß-alanine supplementation on high intensity cycling capacity in normoxia and hypoxia.

The availability of dietary beta-alanine (BA) is the limiting factor in carnosine synthesis within human muscle due to its low intramuscular concentration and substrate affinity. Carnosine can accept hydrogen ions (H+), making it an important intramuscular buffer against exercise-induced acidosis. Metabolite accumulation rate increases when exercising in hypoxic conditions, thus an increased carnosine concentration could attenuate H+ build-up when exercising in hypoxic conditions. This study examined the effects of BA supplementation on high intensity cycling capacity in normoxia and hypoxia. In a double-blind design, nineteen males were matched into a BA group (n = 10; 6.4 g·d-1) or a placebo group (PLA; n = 9) and supplemented for 28 days, carrying out two pre- and two post-supplementation cycling capacity trials at 110% of powermax, one in normoxia and one in hypoxia (15.5% O2). Hypoxia led to a 9.1% reduction in exercise capacity, but BA supplementation had no significant effect on exercise capacity in normoxia or hypoxia (P > 0.05). Blood lactate accumulation showed a significant trial x time interaction post-supplementation (P = 0.016), although this was not significantly different between groups. BA supplementation did not increase high intensity cycling capacity in normoxia, nor did it improve cycling capacity in hypoxia even though exercise capacity was reduced under hypoxic conditions.

Equine metabolism of the selective androgen receptor modulator AC-262536 in vitro and in urine, plasma and hair following oral administration.

AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this study was to investigate the metabolism of AC-262536 in the horse following in vitro incubation and oral administration to two Thoroughbred horses, in order to identify the most appropriate analytical targets for doping control laboratories. Urine, plasma and hair samples were collected and analysed for parent drug and metabolites. Liquid chromatography-high-resolution mass spectrometry was used for in vitro metabolite identification and in urine and plasma samples. Nine phase I metabolites were identified in vitro; four of these were subsequently detected in urine and three in plasma, alongside the parent compound in both matrices. In both urine and plasma samples, the longest detection window was observed for an epimer of the parent compound, which is suggested as the best target for detection of AC-262536 administration. AC-262536 and metabolites were found to be primarily glucuronide conjugates in both urine and plasma. Liquid chromatography-tandem mass spectrometry analysis of post-administration hair samples indicated incorporation of parent AC-262536 into the hair following oral administration. No metabolites were detected in the hair.

Leucine metabolites do not induce changes in phase angle, bioimpedance vector analysis patterns, and strength in resistance trained men.

We aimed to assess the effects of off-the-shelf leucine metabolite supplements on phase angle (PhA), bioimpedance vector analysis (BIVA) patterns and strength during an 8-week resistance training protocol. Fifty-three male participants were allocated into 4 groups: α-hydroxyisocaproic acid (n = 12, age = 30.9 ± 9.3 years), ß-hydroxy-ß-methylbutyrate free acid (n = 12, age = 31.0 ± 9.3 years), calcium ß-hydroxy-ß-methylbutyrate (n = 15, age = 32.1 ± 5.2 years) or placebo (n = 14, age = 28.9 ± 6.6 years). Bioimpedance parameters and 1-repetition maximum (1RM) for back squat and bench press were assessed at baseline and at the end of weeks 4 and 8. Additionally, fat-free mass and fat mass were evaluated by dual-energy X-ray absorptiometry. No statistically group by time interactions were found, even adjusting for age. PhA and vector did not change over the training period, while time-dependent increases were observed for 1RM back squat and 1RM bench press. A direct association was observed between PhA and 1RM bench press changes (whole sample), while PhA and strength were correlated throughout the study, even when adjusting for fat-free mass and percentage of fat mass. Leucine metabolites have no effect on PhA, BIVA patterns or strength during an 8-week resistance training program, in resistance trained subjects. The trial was registered at ClincicalTrials.gov: NCT03511092. Novelty: Supplementation with leucine metabolites is not a supplementation strategy that improves bioelectrical phase angle, cellular health, and strength after an 8-week resistance training program. When consuming a high protein diet, none of the α-hydroxyisocaproic acid, ß-hydroxy-ß-methylbutyrate free acid, and calcium ß-hydroxy-ß-methylbutyrate metabolites resulted in an ergogenic effect in resistance trained men.

Non-carbohydrate Dietary Factors and Their Influence on Post-Exercise Glycogen Storage: a Review.

The optimization of post-exercise glycogen synthesis can improve endurance performance, delay fatigue in subsequent bouts, and accelerate recovery from exercise. High carbohydrate intakes (1.2 g/kg of body weight/h) are recommended in the first 4 h after exercise. However, athletes may struggle to consume carbohydrates at those levels. PURPOSE OF REVIEW: Thus, we aimed to determine whether the consumption of non-carbohydrate dietary factors (creatine, glutamine, caffeine, flavonoids, and alcohol) enhances post-exercise glycogen synthesis. RECENT FINDINGS: Trained athletes may not realize the benefits of creatine loading on glycogen synthesis. The impacts of caffeine, glutamine, flavonoids, and alcohol on post-exercise glycogen synthesis are poorly understood. Other ergogenic benefits to exercise performance, however, have been reported for creatine, glutamine, caffeine, and flavonoids, which were beyond the scope of this review. Evidence in trained athletes is limited and inconclusive on the impact of these non-carbohydrate dietary factors on post-exercise glycogen synthesis.

Re-esterified DHA improves ventilatory threshold 2 in competitive amateur cyclists.

BACKGROUND: Fish oils were studied as ergogenic aids in a number of mixed physical trial designs showing promising results. However, the heterogeneous purity of the studied supplements, combined with the variety of physical tests employed call for more studies to confirm these findings, ideally with standardised supplements. Our aim was to test a supplement highly concentrated in DHA (DHA:EPA ratio equal to approximately 8:1) on a maximal cycling test to elucidate performance improvements mainly due to DHA. METHODS: A double-blind, placebo controlled, randomised balanced, parallel design, in competitive amateur cyclists was employed. They were all male, older than 18 years old, with training routine of 2 to 4 sessions per week lasting at least one hour each. A ramp cycling test to exhaustion with a subsequent 5 min recovery phase was employed before and after treatment to analyse aerobic metabolism and lactate clearance after the bout. After 30 days of supplementation with 975 mg of re-esterified DHA, the thirty-eight cyclist who completed the study were finally included for statistical analysis. RESULTS: Mean power output at ventilatory threshold 2 (VT2) improved after DHA supplementation both as absolute (△DHA versus △PLA: 6.33-26.54 Watts; CI 95%) and relative (p=0.006) values, paralleled with higher oxygen consumption at VT2 both for absolute (DHA 2729.4 ±304.5, 3045.9 ±335.0; PLA 2792.3 ±339.5, 2845.5 ±357.1; ml·min-1 baseline versus post p=0.025) and relative values (DHA 36.6 ±5.0, 41.2 ±5.4; PLA 37.2 ±5.7, 38.1 ±5.2; ml·kg-1·min-1 baseline versus post p=0.024). Heart rate recovery rate improved during the recovery phase in the DHA group compared to PLA (p=0.005). CONCLUSION: DHA is capable of improving mean power output at the ventilatory threshold 2 (anaerobic ventilatory threshold) in amateur competitive cyclists. It is unclear if these findings are the result of the specific DHA supplement blend or another factor.

Investigation of the metabolism of the selective androgen receptor modulator LGD-4033 in equine urine, plasma and hair following oral administration.

LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral administration, in order to identify the most appropriate analytical targets for doping control laboratories. LGD-4033 was orally administered to two Thoroughbred horses and urine, plasma and hair samples were collected and analysed for parent drug and metabolites. LC-HRMS was used for metabolite identification in urine and plasma. Eight metabolites were detected in urine, five of which were excreted only as phase II conjugates, with the longest detection time being observed for di- and tri-hydroxylated metabolites. The parent compound could only be detected in urine in the conjugated fraction. Seven metabolites were detected in plasma along with the parent compound where mono-hydroxylated metabolites provided the longest duration of detection. Preliminary investigations with hair samples using LC-MS/MS analysis indicated the presence of trace amounts of the parent compound and one of the mono-hydroxylated metabolites. In vitro incubation of LGD-4033 with equine liver microsomes was also performed for comparison, yielding 11 phase I metabolites. All of the metabolites observed in vivo were also observed in vitro.

Coffee Ingestion Improves 5 km Cycling Performance in Men and Women by a Similar Magnitude.

Caffeine is a well-established ergogenic aid, although research to date has predominantly focused on anhydrous caffeine, and in men. The primary aim of the present study was to investigate the effect of coffee ingestion on 5 km cycling time trial performance, and to establish whether sex differences exist. A total of 38 participants (19 men and 19 women) completed a 5 km time trial following the ingestion of 0.09 g·kg-1 coffee providing 3 mg·kg-1 of caffeine (COF), a placebo (PLA), in 300 mL of water, or control (CON). Coffee ingestion significantly increased salivary caffeine levels (p < 0.001; η P 2 = 0.75) and, overall, resulted in improved 5 km time trial performance (p < 0.001; η P 2 = 0.23). Performance following COF (482 ± 51 s) was faster than PLA (491 ± 53 s; p = 0.002; d = 0.17) and CON (487 ± 52 s; p =0.002; d = 0.10) trials, with men and women both improving by approximately 9 seconds and 6 seconds following coffee ingestion compared with placebo and control, respectively. However, no differences were observed between CON and PLA (p = 0.321; d = 0.08). In conclusion, ingesting coffee providing 3 mg·kg-1 of caffeine increased salivary caffeine levels and improved 5 km cycling time trial performance in men and women by a similar magnitude.

No ergogenic effects of a 10-day combined heat and hypoxic acclimation on aerobic performance in normoxic thermoneutral or hot conditions.

PURPOSE: Hypoxic acclimation enhances convective oxygen delivery to the muscles. Heat acclimation-elicited thermoregulatory benefits have been suggested not to be negated by adding daily exposure to hypoxia. Whether concomitant acclimation to both heat and hypoxia offers a synergistic enhancement of aerobic performance in thermoneutral or hot conditions remains unresolved. METHODS: Eight young males ([Formula: see text]: 51.6 ± 4.6 mL min-1 kg-1) underwent a 10-day normobaric hypoxic confinement (FiO2 = 0.14) interspersed with daily 90-min normoxic controlled hyperthermia (target rectal temperature: 38.5 °C) exercise sessions. Prior to, and following the confinement, the participants conducted a 30-min steady-state exercise followed by incremental exercise to exhaustion on a cycle ergometer in thermoneutral normoxic (NOR), thermoneutral hypoxic (FiO2 = 0.14; HYP) and hot (35 °C, 50% relative humidity; HE) conditions in a randomized and counterbalanced order. The steady-state exercise was performed at 40% NOR peak power output (Wpeak) to evaluate thermoregulatory function. Blood samples were obtained from an antecubital vein before, on days 1 and 10, and the first day post-acclimation. RESULTS: [Formula: see text] and ventilatory thresholds were not modified in any environment following acclimation. Wpeak increased by 6.3 ± 3.4% in NOR and 4.0 ± 4.9% in HE, respectively. The magnitude and gain of the forehead sweating response were augmented in HE post-acclimation. EPO increased from baseline (17.8 ± 7.0 mIU mL-1) by 10.7 ± 8.8 mIU mL-1 on day 1 but returned to baseline levels by day 10 (15.7 ± 5.9 mIU mL-1). DISCUSSION: A 10-day combined heat and hypoxic acclimation conferred only minor benefits in aerobic performance and thermoregulation in thermoneutral or hot conditions. Thus, adoption of such a protocol does not seem warranted.