Effect of repeated prolonged exercise on liver fat content and visceral adipose tissue in well-trained older men.

INTRODUCTION: Liver fat (LF) and visceral adipose tissue (VAT) content decreases with training, however, this has mainly been investigated in sedentary obese or healthy participants. The aim of this study was to investigate the effects of repeated prolonged exercise on LF and VAT content in well-trained older men and to compare baseline LF and VAT content to recreationally active older men. METHOD: A group of five well-trained older men were tested before and after cycling a total distance of 2558 km in 16 consecutive days. VAT content and body composition was measured using DXA before a bicycle ergometer test was performed to determine maximal fat oxidation (MFO), maximal oxygen consumption ( VO 2 max $$ {\mathrm{VO}}_{2_{\mathrm{max}}} $$ ), and the relative intensity at which MFO occurred (Fatmax). LF content was measured on a separate day using MRI. For comparison of baseline values, a control group of eight healthy age- and BMI-matched recreationally active men were recruited. RESULTS: The well-trained older men had lower VAT (p = 0.02), and a tendency toward lower LF content (p = 0.06) compared with the control group. The intervention resulted in decreased LF content (p = 0.02), but VAT, fat mass, and lean mass remained unchanged. VO 2 max $$ {\mathrm{VO}}_{2_{\mathrm{max}}} $$ , MFO, and Fatmax were not affected by the intervention. CONCLUSION: The study found that repeated prolonged exercise reduced LF content, but VAT and VO 2 max $$ {\mathrm{VO}}_{2_{\mathrm{max}}} $$ remained unchanged. Aerobic capacity was aligned with lower LF and VAT in older active men.

Prolonged Endurance Exercise Increases Macrophage Content and Mitochondrial Respiration in Adipose Tissue in Trained Men.

BACKGROUND: The aim of this study was to investigate the effect of prolonged endurance exercise on adipose tissue inflammation markers and mitochondrial respiration in younger and older men. METHODS: "Young" (aged 30 years, n = 7) and "old" (aged 65 years, n = 7) trained men were exposed to an exercise intervention of 15 consecutive days biking 7 to 9 hours/day at 63% and 65% of maximal heart rate (young and old, respectively), going from Copenhagen, Denmark to Palermo, Italy. Adipose tissue was sampled from both the gluteal and abdominal depot before and after the intervention. Mitochondrial respiration was measured by high-resolution respirometry, and adipose inflammation was assessed by immunohistochemical staining of paraffin embedded sections. RESULTS: An increased number of CD163+ macrophages was observed in both the gluteal and abdominal depot (P < .01). In addition, an increased mitochondrial respiration was observed in the abdominal adipose tissue from men in the young group with complex I (CIp) stimulated respiration, complex I + II (CI+IIp) stimulated respiration and the capacity of the electron transport system (ETS) (P < .05), and in the older group an increase in CIp and CI+IIp stimulated respiration (P < .05) was found. CONCLUSION: Overall, we found a positive effect of prolonged endurance exercise on adipose tissue inflammation markers and mitochondrial respiration in both young and old trained men, and no sign of attenuated function in adipose tissue with age.

Physical activity impacts resting skeletal muscle myosin conformation and lowers its ATP consumption.

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as the super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize ATP consumption and skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 5- to 10-fold reduction in ATP turnover compared with DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibers from young men of various physical activity levels (sedentary, moderately physically active, endurance-trained, and strength-trained athletes) and ran a loaded Mant-ATP chase protocol. We observed that in moderately physically active individuals, the amount of myosin molecules in the SRX state in type II muscle fibers was significantly greater than in age-matched sedentary individuals. In parallel, we did not find any difference in the proportions of SRX and DRX myosins in myofibers between highly endurance- and strength-trained athletes. We did however observe changes in their ATP turnover time. Altogether, these results indicate that physical activity level and training type can influence the resting skeletal muscle myosin dynamics. Our findings also emphasize that environmental stimuli such as exercise have the potential to rewire the molecular metabolism of human skeletal muscle through myosin.

Extreme duration exercise affects old and younger men differently.

AIM & METHODS: Extreme endurance exercise provides a valuable research model for understanding the adaptive metabolic response of older and younger individuals to intense physical activity. Here, we compare a wide range of metabolic and physiologic parameters in two cohorts of seven trained men, age 30 ± 5 years or age 65 ± 6 years, before and after the participants travelled ≈3000 km by bicycle over 15 days. RESULTS: Over the 15-day exercise intervention, participants lost 2-3 kg fat mass with no significant change in body weight. V̇O2 max did not change in younger cyclists, but decreased (p = 0.06) in the older cohort. The resting plasma FFA concentration decreased markedly in both groups, and plasma glucose increased in the younger group. In the older cohort, plasma LDL-cholesterol and plasma triglyceride decreased. In skeletal muscle, fat transporters CD36 and FABPm remained unchanged. The glucose handling proteins GLUT4 and SNAP23 increased in both groups. Mitochondrial ROS production decreased in both groups, and ADP sensitivity increased in skeletal muscle in the older but not in the younger cohort. CONCLUSION: In summary, these data suggest that older but not younger individuals experience a negative adaptive response affecting cardiovascular function in response to extreme endurance exercise, while a positive response to the same exercise intervention is observed in peripheral tissues in younger and older men. The results also suggest that the adaptive thresholds differ in younger and old men, and this difference primarily affects central cardiovascular functions in older men after extreme endurance exercise.

The effect of 8 weeks of physical training on muscle performance and maximal fat oxidation rates in patients treated with simvastatin and coenzyme Q10 supplementation.

Statins are prescribed for the treatment of elevated cholesterol, but they may negatively affect metabolism, muscle performance, and the response to training. Coenzyme Q10 (CoQ10) supplementation may alleviate these effects. Combined simvastatin and CoQ10 treatment during physical training has never been tested. We studied the response to 8 weeks training (maximal oxygen uptake ( V̇O2max${\dot{V}_{{{\rm{O}}_{\rm{2}}}{\rm{max}}}}$ ), fat oxidation (MFO), the workload at which MFO occurred, and muscle strength) in statin naive dyslipidaemic patients who received simvastatin (40 mg/day) with (S + Q, n = 9) or without (S + Pl, n = 10) CoQ10 supplementation (2 × 200 mg/day) or placebo (Pl + Pl, n = 7) in a randomized, double-blind placebo-controlled study. V̇O2max${\dot{V}_{{{\rm{O}}_{\rm{2}}}{\rm{max}}}}$ and maximal workload increased with training (main effect of time, P < 0.05). MFO increased from 0.29 ± 0.10, 0.26 ± 0.10, and 0.38 ± 0.09 to 0.42 ± 0.09, 0.38 ± 0.10 and 0.48 ± 0.16 g/min in S + Q, S + Pl, and Pl + Pl, respectively (main effect of time, P = 0.0013). The workload at MFO increased from 75 ± 25, 56 ± 23, and 72 ± 17 to 106 ± 25, 84 ± 13 and 102 ± 31 W in S + Q, S + Pl, and Pl + Pl, respectively (main effect of time, P < 0.0001). Maximal voluntary contraction and rate of force development were unchanged. Exercise improved aerobic physical capacity and simvastatin with or without CoQ10 supplementation did not inhibit this adaptation. The similar increases in MFO and in the workload at which MFO occurred in response to training shows that the ability to adapt substrate selection and oxidation rates is preserved with simvastatin treatment, despite the potential negative impact of simvastatin at the mitochondrial level. CoQ10 supplementation does not augment this adaptation. KEY POINTS: Simvastatins are prescribed for treatment of elevated cholesterol, but they may negatively affect metabolism, muscle performance and the response to training. Coenzyme Q10 (CoQ10) supplementation may alleviate some of these effects. We found that simvastatin treatment does not negatively affect training-induced adaptations of substrate oxidation during exercise. Likewise, maximal oxygen uptake increases with physical training also in patients in treatment with simvastatin. CoQ10 supplementation in simvastatin-treated patients presents no advantage in the adaptations to physical training Simvastatin treatment decreases plasma concentrations of total CoQ10, but this can be alleviated by simultaneous supplementation with CoQ10.

Acute erythropoietin injection increases muscle mitochondrial respiratory capacity in young men: a double-blinded randomized crossover trial.

The aim was to investigate if acute recombinant human erythropoietin (rHuEPO) injection had an effect on mitochondrial function and if exercise would have an additive effect. Furthermore, to investigate if in vitro incubation with rHuEPO had an effect on muscle mitochondrial respiratory capacity. Eight healthy young men were recruited for this double-blinded randomized placebo-controlled crossover study. rHuEPO (400 IU/kg body wt) or saline injection was given intravenously, before an acute bout of exercise. Resting metabolic rate and fat oxidation were measured. Biopsies were obtained at baseline, 120 min after injection, and right after the acute exercise bout. Mitochondrial function (mitochondrial respiration and H2O2 emission) was measured in permeabilized skeletal muscle using high-resolution respirometry and fluorometry. Specific gene expression and enzyme activity were measured. Skeletal muscle mitochondrial respiratory capacity was measured with and without incubation with rHuEPO. Fat oxidation at rest increased after rHuEPO injection, but no difference was found in fat oxidation during exercise. Mitochondrial respiratory capacity was increased after rHuEPO injection when pyruvate was in the assay, which was not the case when saline was injected. No changes were seen in H2O2 emission after rHuEPO injection or acute exercise. Incubation of skeletal muscle fibers in vitro with rHuEPO increased mitochondrial respiratory capacity. Acute rHuEPO injection increased mitochondrial respiratory capacity when pyruvate was used in the assay. No statistical difference was found in H2O2 emission capacity, although a numerical increase was seen after rHuEPO injection. In vitro incubation of the skeletal muscle sample with rHuEPO increases mitochondrial respiratory capacity.NEW & NOTEWORTHY The effect of an acute rHuEPO injection on skeletal muscle mitochondrial function was investigated in young healthy male subjects. rHuEPO has an acute effect on skeletal muscle mitochondrial respiratory capacity in humans, where an increased mitochondrial respiratory capacity was seen. This could be the first step leading to increased mitochondrial biogenesis.

Peak Fat Oxidation Rate Is Closely Associated With Plasma Free Fatty Acid Concentrations in Women; Similar to Men.

Introduction: In men, whole body peak fat oxidation (PFO) determined by a graded exercise test is closely tied to plasma free fatty acid (FFA) availability. Men and women exhibit divergent metabolic responses to fasting and exercise, and it remains unknown how the combined fasting and exercise affect substrate utilization in women. We aimed to investigate this, hypothesizing that increased plasma FFA concentrations in women caused by fasting and repeated exercise will increase PFO during exercise. Then, that PFO would be higher in women compared with men (data from a previous study). Methods: On two separate days, 11 young endurance-trained women were investigated, either after an overnight fast (Fast) or 3.5 h after a standardized meal (Fed). On each day, a validated graded exercise protocol (GXT), used to establish PFO by indirect calorimetry, was performed four times separated by 3.5 h of bed rest both in the fasted (Fast) or fed (Fed) state. Results: Peak fat oxidation increased in the fasted state from 11 ± 3 (after an overnight fast, Fast 1) to 16 ± 3 (mean ± SD) mg/min/kg lean body mass (LBM) (after ~22 h fast, Fast 4), and this was highly associated with plasma FFA concentrations, which increased from 404 ± 203 (Fast 1) to 865 ± 210 µmol/L (Fast 4). No increase in PFO was found during the fed condition with repeated exercise. Compared with trained men from a former identical study, we found no sex differences in relative PFO (mg/min/kg LBM) between men and women, in spite of significant differences in plasma FFA concentrations during exercise after fasting. Conclusion: Peak fat oxidation increased with fasting and repeated exercise in trained women, but the relative PFO was similar in young trained men and women, despite major differences in plasma lipid concentrations during graded exercise.

Maximal Fat Oxidation Rate Is Higher in Fit Women and Unfit Women With Obesity, Compared to Normal-weight Unfit Women.

CONTEXT: The maximal fat oxidation rate (MFO) is higher in aerobically fit vs unfit young men, but this training-related increase in MFO is attenuated in middle-aged men. Further, it has also been found that unfit men with obesity may have an elevated MFO compared to unfit normal-weight men. OBJECTIVE: Based hereupon, we aimed to investigate whether a fitness-related higher MFO were attenuated in middle-aged women compared to young women. Also, we aimed to investigate if unfit women with obesity have a higher MFO compared to unfit normal-weight women. We hypothesized that the training-related elevated MFO was attenuated in middle-aged women, but that unfit women with obesity would have an elevated MFO compared to unfit normal-weight women. METHODS: We recruited 70 women stratified into 6 groups: young fit (n = 12), young unfit (n = 12) middle-aged fit (n = 12), middle-aged unfit (n = 12), unfit young women with obesity (n = 12), and unfit middle-aged women with obesity (n = 10). Body composition and resting blood samples were obtained and MFO was measured by a graded exercise test on a cycle ergometer via indirect calorimetry. Subsequently, a maximal exercise test was performed to establish peak oxygen uptake (V̇O2peak). RESULTS: Young and middle-aged fit women had a higher MFO compared to age-matched unfit women, and young fit women had a higher MFO compared to fit middle-aged women. Unfit women with obesity, independent of age, had a higher MFO compared to their normal-weight and unfit counterparts. CONCLUSION: The training-related increase in MFO seems maintained in middle-aged women, and we find that unfit women with obesity, independent of age, have a higher MFO compared to unfit normal-weight women.

The training induced increase in whole-body peak fat oxidation rate may be attenuated with aging.

An attenuated ability to appropriately oxidize fat (metabolic inflexibility) has been associated with the development of obesity and type 2 diabetes. Previous studies have found that regular exercise training increases the body's ability to oxidize fat during exercise, but also shown that fat oxidation at the same relative and absolute exercise intensity is lower in old compared with young adults. Based on these studies we investigated the effect of training status on the whole-body peak fat oxidation rate (PFO) during exercise in young and middle-aged trained and untrained men. We hypothesized that aging was associated with decreased PFO, but regular exercise training could counteract this decline. 36 healthy non-overweight young and middle-aged men were recruited into a four groups: young (27 [24-30] yrs, (Mean [95% CI])) untrained (⩒O2peak: 47 [44-49] ml/min/kg), young (28 [26-30] yrs) trained (⩒O2peak: 64 [62-67] ml/min/kg), middle-aged (55 [53-57] yrs) untrained (⩒O2peak: 37 [32-42] ml/min/kg) and middle-aged (54 [51-57] yrs) trained (⩒O2peak: 55 [51-58] ml/min/kg). PFO was measured by indirect calorimetry while subjects performed a validated incremental exercise protocol on a cycle ergometer. Whole-body peak fat oxidation rate was higher in the young trained compared to young untrained subjects (0.70 [0.65-0.75] vs.0.45 [0.36-0.54] g/min, post-hoc: p < 0.001); however, this training effect was attenuated in middle-aged trained and untrained subjects (0.44 [0.38-0.50] vs. 0.41 [0.35-0.47] g/min, post-hoc: p = 0.83, respectively). In summary, these findings suggest that the training induced effects on whole-body fat oxidation found in young men may be attenuated in middle-aged men.

The relationship between peak fat oxidation and prolonged double-poling endurance exercise performance.

The peak fat oxidation rate (PFO) and the exercise intensity that elicits PFO (Fatmax ) are associated with endurance performance during exercise primarily involving lower body musculature, but it remains elusive whether these associations are present during predominant upper body exercise. The aim was to investigate the relationship between PFO and Fatmax determined during a graded exercise test on a ski-ergometer using double-poling (GET-DP) and performance in the long-distance cross-country skiing race, Vasaloppet. Forty-three healthy men completed GET-DP and Vasaloppet and were divided into two subgroups: recreational (RS, n = 35) and elite (ES, n = 8) skiers. Additionally, RS completed a cycle-ergometer GET (GET-Cycling) to elucidate whether the potential relationships were specific to exercise modality. PFO (r2  = .10, P = .044) and Fatmax (r2  = .26, P < .001) were correlated with performance; however, V ˙ O 2 peak was the only independent predictor of performance (adj. R2  = .36) across all participants. In ES, Fatmax was the only variable associated with performance (r2  = .54, P = .038). Within RS, DP V ˙ O 2 peak (r2  = .11, P = .047) and ski-specific training background (r2  = .30, P = .001) were associated with performance. Between the two GETs, Fatmax (r2  = .20, P = .006) but not PFO (r2  = .07, P = .135) was correlated. Independent of exercise mode, neither PFO nor Fatmax were associated with performance in RS (P > .05). These findings suggest that prolonged endurance performance is related to PFO and Fatmax but foremost to V ˙ O 2 peak during predominant upper body exercise. Interestingly, Fatmax may be an important determinant of performance among ES. Among RS, DP V ˙ O 2 peak , and skiing experience appeared as performance predictors. Additionally, whole-body fat oxidation seemed specifically coupled to exercise modality.

Menstrual cycle phase does not affect whole body peak fat oxidation rate during a graded exercise test.

Female sex hormones fluctuate in a predictable manner throughout the menstrual cycle in eumenorrheic women. In studies conducted in both animal and humans, estrogen and progesterone have been found to exert individual metabolic effects during both rest and exercise, suggesting that estrogen may cause an increase in fat oxidation during exercise. However, not all studies find these metabolic changes with the natural physiological variation in the sex hormones. To date, no studies have investigated whether whole body peak fat oxidation rate (PFO) and maximal fat oxidation intensity (FATmax) are affected at different time points [mid-follicular (MF), late-follicular (LF), and mid-luteal (ML)] in the menstrual cycle, where plasma estrogen and progesterone are either at their minimum or maximum. We hypothesized that an increased plasma estrogen concentration together with low progesterone concentration in LF would result in a modest but significant increase in PFO. We found no differences in body weight, body composition, or peak oxygen uptake (V̇o2peak) between any of the menstrual phases in the 19 healthy, young eumenorrheic women included in this study. PFO [MF: 0.379 (0.324-0.433) g/min; LF: 0.375 (0.329-0.421) g/min; ML: 0.382 (0.337-0.442) g/min; mean ± (95% CI)] and resting plasma free fatty acid concentrations [MF: 392 (293-492) µmol/l; LF: 477 (324-631) µmol/l; ML: 396 (285-508) µmol/L] were also similar across the menstrual cycle phases. Contrary to our hypothesis, we conclude that the naturally occurring fluctuations in the sex hormones estrogen and progesterone do not affect the whole body PFO and FATmax in young eumenorrheic women measured during a graded exercise test.NEW & NOTEWORTHY Menstrual cycle phase does not affect the peak fat oxidation rate during a graded exercise test. Natural physiological fluctuations in estrogen do not increase peak fat oxidation rate. FATmax is not influenced by menstrual cycle phase in healthy, young eumenorrheic women.

Determination and validation of peak fat oxidation in endurance-trained men using an upper body graded exercise test.

Peak fat oxidation rate (PFO) and the intensity that elicits PFO (Fatmax ) are commonly determined by a validated graded exercise test (GE) on a cycling ergometer with indirect calorimetry. However, for upper body exercise fat oxidation rates are not well elucidated and no protocol has been validated. Thus, our aim was to test validity and inter-method reliability for determination of PFO and Fatmax in trained men using a GE protocol applying double poling on a ski-ergometer. PFO and Fatmax were assessed during two identical GE tests (GE1 and GE2) and validated against separated short continuous exercise bouts (SCE) at 35%, 50%, and 65% of V̇O2peak on the ski-ergometer in 10 endurance-trained men (V̇O2peak : 65.1 ± 1.0 mL·min-1 ·kg-1 , mean ± SEM). Between GE tests no differences were found in PFO (GE1: 0.42 ± 0.03; GE2: 0.45 ± 0.03 g·min-1 , P = .256) or Fatmax (GE1: 41 ± 2%; GE2: 43 ± 3% of V̇O2peak , P = .457) and the intra-individual coefficient of variation (CV) was 8 ± 2% and 11 ± 2% for PFO and Fatmax , respectively. Between GE and SCE tests, PFO (GEavg : 0.44 ± 0.03; SCE; 0.47 ± 0.06 g·min-1 , P = .510) was not different, whereas a difference in Fatmax (GEavg : 42 ± 2%; SCE: 52 ± 4% of V̇O2peak , P = .030) was observed with a CV of 17 ± 4% and 15 ± 4% for PFO and Fatmax , respectively. In conclusion, GE has a high day-to-day reliability in determination of PFO and Fatmax in trained men, whereas it is unclear if PFO and Fatmax determined by GE reflect continuous exercise in general.

Plasma free fatty acid concentration is closely tied to whole body peak fat oxidation rate during repeated exercise.

Plasma free fatty acids (FFA) are a major contributor to whole body fat oxidation during exercise. However, the extent to which manipulating plasma FFA concentrations will influence whole body peak fat oxidation rate (PFO) during exercise remains elusive. In this study we aimed to increase plasma FFA concentrations through a combination of fasting and repeated exercise bouts. We hypothesized that an increase in plasma FFA concentration would increase PFO in a dose-dependent manner. Ten healthy young (31 ± 6 yr) (mean ± SD) well-trained (maximal oxygen uptake 65.9 ± 6.1 ml·min-1·kg-1) men performed four graded exercise tests (GXTs) on 1 day. The GXTs were interspersed by 4 h of bed rest. This was conducted either in a fasted state or with the consumption of a standardized carbohydrate-rich meal 3.5 h before each GXT. Fasting and previous GXTs resulted in a gradual increase in PFO from 0.63 ± 0.18 g/min after an overnight fast (10 h) to 0.93 ± 0.17 g/min after ∼22 h of fasting and three previous GXTs. This increase in PFO coincided with an increase in plasma FFA concentrations (r2 = 0.73, P < 0.0001). Ingestion of a carbohydrate-rich meal 3.5 h before each GXT resulted in unaltered PFO. This was also reflected in unchanged plasma FFA, glucose, and insulin concentrations. In this study we show that plasma FFA availability is closely tied to whole body PFO and that the length of fasting combined with previous exercise are robust stimuli toward increasing plasma FFA concentration, highlighting the importance for preexercise standardization when conducting GXTs measuring substrate oxidation. NEW & NOTEWORTHY We show that peak fat oxidation is increased in close relationship with plasma free fatty acid availability after combined fasting and repeated incremental exercise tests in healthy highly trained men. Therefore it may be argued that whole body fat oxidation rate measured in most cases after an overnight fast indeed does not represent whole body maximal fat oxidation rate but a whole body peak fat oxidation rate within the context of the preexercise standardization obtained in the study design.

Peak Fat Oxidation is not Independently Related to Ironman Performance in Women.

The aim of the present study was to investigate if peak fat oxidation rate (PFO) is related to Ironman performance in female athletes. Thirty-six female Ironman athletes (age: 34±1 yrs, [21-45 yrs.] SEM [Range]) with a BMI of 22.1±2.0 kg/m2 [18.8-28.4 kg/m2], a body fat percentage of 24.8±1.0% [9.0-37.0%] and a V̇O2peak of 53.0±1.3 ml/min/kg [36.5-70.5 ml/min/kg] were tested in the laboratory prior to the Ironman Copenhagen 2017. Race time ranged from 9:17:07 to 15:23:48 with mean race time being 11:57:26 h:min:s (717 min). By simple linear regression analyses we found associations between race time and P FO (r2=0.22, p<0.005), V̇O2peak (r2=0.65, p<0.0001) and the relative exercise intensity eliciting PFO (Fatmax) (r2=0.35, p=0.0001). Furthermore, associations were found between race time and body fat percentage (r2=0.44, p<0.0001) and age (r2=0.16, p<0.05). By means of multiple regression analysis, V̇O2peak was the only statistically significant variable explaining 64% of the variation in race time (adj. r2=0.64, p<0.005). In conclusion, these results demonstrate that PFO is not independently related to Ironman performance in a heterogeneous group of female athletes. Interestingly, V̇O2peak alone was able to predict 64% of the variation in Ironman race times.

Maximal Fat Oxidation is Related to Performance in an Ironman Triathlon.

The aim of the present study was to investigate the relationship between maximal fat oxidation rate (MFO) measured during a progressive exercise test on a cycle ergometer and ultra-endurance performance. 61 male ironman athletes (age: 35±1 yrs. [23-47 yrs.], with a BMI of 23.6±0.3 kg/m2 [20.0-30.1 kg/m2], a body fat percentage of 16.7±0.7% [8.4-30.7%] and a VO2peak of 58.7±0.7 ml/min/kg [43.9-72.5 ml/min/kg] SEM [Range]) were tested in the laboratory between 25 and 4 days prior to the ultra-endurance event, 2016 Ironman Copenhagen. Simple bivariate analyses revealed significant negative correlations between race time and MFO (r2=0.12, p<0.005) and VO2peak (r2=0.45, p<0.0001) and a positive correlation between race time and body fat percentage (r2=0.27, p<0.0001). MFO and VO2peak were not correlated. When the significant variables from the bivariate regression analyses were entered into the multiple regression models, VO2peak and MFO together explained 50% of the variation observed in race time among the 61 Ironman athletes (adj R2=0.50, p<0.001). These results suggests that maximal fat oxidation rate exert an independent influence on ultra-endurance performance (>9 h). Furthermore, we demonstrate that 50% of the variation in Ironman triathlon race time can be explained by peak oxygen uptake and maximal fat oxidation.