The aim of this study was to evaluate time-of-day effects on fatigue during a sustained anaerobic cycling exercise. Sixteen healthy male competitive cyclists were asked to perform a 60 s Wingate test against a braking load of 0.087 kg.kg body mass(-1) during two experimental sessions, which were set up either at 06:00 or 18:00 h in counterbalanced order. There was only one session per day with a recovery period of at least 36 h between the two sessions. Each subject was trained to perform the test. The body mass used to determine the braking load was that of the first test session for each subject and remained constant throughout the two test periods. During the test, peak power (PP), mean power during the first 30 s (MP30 s) and the full 60 s of the test (MP60 s), and fatigue (i.e., the decrease in power output values throughout the exercise) were analyzed. Results confirmed the existence of diurnal variation in anaerobic power output. PP, MP30 s, and MP60 s were significantly higher at 18:00 than 06:00 h, with gains equal to 8.2, 7.8, and 7.8%, respectively. Moreover, all the power output values recorded in the evening were higher than those recorded in the morning, indicating that fatigue induced by this exercise is not affected by time-of-day in male competitive cyclists. It is hypothesized that the freedom and complexity of pedalling could allow adaptations in movement patterns, as a function of time-of-day, in order to maintain higher performance in the evening. For practical considerations, the more complex the movements required to perform a sport, the more the time-of-day effect can be taken into account and adapted to by the trained athlete, particularly in cyclic sporting disciplines such as swimming, running, rowing, and kayaking.