The pungent bioactive ingredient in peppers (Capsaicin; CAP) has been widely investigated for its activation of the transient receptor potential vanilloid 1 (TRPV1) channel on sensory neurons. It has been shown to improve mitochondrial biogenesis, adenosine triphosphate (ATP) production and vascular function. In animal models has been demonstrated that increases exercise performance and decreases fatigue development. However, whether acute oral CAP improves endurance performance and the potential underlying mechanisms remains elusive. This study aimed to evaluate the potential ergogenic and anti-fatigue effects of acute oral CAP supplementation in healthy humans. In a double-blind, placebo-controlled, cross-over design, 10 young healthy males performed constant-load cycling exercise (294±55W; 85% of peak power output) to exhaustion, after acute ingestion of placebo (PL; 1000mg fiber pill) and CAP (780mg pill) capsules. Fatigue was assessed on the dominant quadriceps with the interpolated twitch technique, via changes in supramaximal electrical stimulation pre- vs. post-exercise, during isometric maximal voluntary contractions (MVC) and at rest. Cardiopulmonary parameters (heart rate, cardiac output, oxygen consumption, ventilation) were assessed throughout the exercise. No statistically significant differences were detected in the muscle contractile functions, despite a trend (P=0.07) for a decreased time to exhaustion during CAP (PL: 369±85 vs. CAP: 296±64 sec). All the values (except muscle voluntary activation (%VMA)) significantly decreased from pre- to post-exercise in both conditions. The exercise-induced attenuation of the intrinsic muscle contractile properties (Maximal rate of force development (MRFD) and Maximal relaxation rate (MRR) of the resting twitch force) were mitigated, in part, by CAP. Notably, in the PL condition MRR was seemingly reduced by 47±33%, while only of 29±68% in CAP; contrarily, MRFD decreased similarly of 47±23% and of 40±27% in PL and CAP, respectively. In 9 participants, the resting twitch (estimate of peripheral muscle fatigue) pre- vs. post-exercise decreased less with CAP (PL: 44±17 vs. CAP: 37±13%). CAP did not influence the MVC nor %VMA. The cardiopulmonary responses were similar between the conditions (P>0.05). In contrast to prior work, acute oral capsaicin did not improve endurance performance in the present study. CAP did elicit a tendency for faster relaxation rate, which might suggest an enhanced activation of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumps, thereby increasing the rate of Ca2+ reuptake and relaxation. However, this altered Ca2+ handling might also increase ATP demand. The tendency for CAP to attenuate the exercise-induced reduction in muscle function, or fatigue, might provide a mechanistic basis for the previously demonstrated beneficial effects of CAP. Further work is needed in larger samples, to further determine the optimal dosing and loading strategies, to better understand the potential ergogenic effects of capsaicin.