The present study was designed to evaluate time-of-day effects on electromyographic (EMG) activity changes during a short-term intense cycling exercise. In a randomized order, 22 male subjects were asked to perform a 30-s Wingate test against a constant braking load of 0.087 kg·kg(-1) body mass during two experimental sessions, which were set up either at 07:00 or 17:00 h. During the test, peak power (P(peak)), mean power (P(mean)), fatigue index (FI; % of decrease in power output throughout the 30 s), and evolution of power output (5-s span) throughout the exercise were analyzed. Surface EMG activity was recorded in both the vastus lateralis and vastus medialis muscles throughout the test and analyzed over a 5-s span. The root mean square (RMS) and mean power frequency (MPF) of EMG were calculated. Neuromuscular efficiency (NME) was estimated from the ratio of power to RMS. Resting core temperature, P(peak), P(mean), and FI were significantly higher (p < .05) in the evening than morning test (e.g., P(peak): 11.6 ± 0.8 vs. 11.9 ± 1 W·kg(-1)). The results showed that power output decreased following two phases. During the first phase (first 20s), power output decreased rapidly and values were higher (p < .05) in the evening than in the morning. During the second phase (last 10s), power decreased slightly and appeared independent of the time of day of testing. This power output decrease was paralleled by evolution of the MPF and NME. During the first phase, NME and MPF were higher (p < .05) in the evening. During the second phase, NME and MPF were independent of time of day. In addition, no significant differences were noticed between 7:00 and 17:00 h for EMG RMS during the whole 30 s. Taken together, these results suggest that peripheral mechanisms (i.e., muscle power and fatigue) are more likely the cause of the diurnal variation of the Wingate-test performance rather than central mechanisms.