The purpose of this investigation was to assess the effects of rest interval following active warm-up (WU) durations on the diurnal variation of high-intensity cycling performance. Eleven male physical education students (22.6 ± 2.5 years; 179.2 ± 5.7 cm; 82.6 ± 9.6 kg; mean ± SD) participated in a cross-over randomized study, and they all underwent the 30-s Wingate test in the morning (08:00 h) and in the evening (18:00 h), after 5-min (WU 5 ) and 15-min (WU 15 ) warm-up durations, either with rest (WR), or without rest interval (NR) separating the WU at the onset of the high-intensity cycling exercise performance. The WU consisted of pedaling at a constant pace of 60 rpm against at 50% of the maximal aerobic power. The rest interval between the end of warm-up and the beginning of the anaerobic exercise was set at 5 min. Peak power (PP), mean power (MP), and the fatigue index (FI) were recorded. Likewise, heart rate, oral temperature (T), and rating of perceived exertion were registered at rest, at the end of the WU and just after the Wingate test. The ANOVA's showed no main effect of the rest interval on PP, MP, FI, and T parameters. However, significant interactions (WU duration × time-of-day and recovery condition × WU duration) were recorded on both PP and MP parameters. PP and MP were higher in the afternoon compared to the morning with gains of 4.4 and 3.6%, respectively. In the morning sessions, the WU 15 allows better improvement of muscular power, with either 0-or 5-min pre-exercise rest interval. However, in the afternoon sessions, both WU 15 and WU 5 durations allow better improvement of 30-s Wingate cycling performance in, respectively, WR and NR conditions. Therefore, athletes and coaches, as well as researchers, interested in high-intensity cycling exercise, should take into account the rest interval, the time-of-day, and the duration of warm-up when practicing, assessing, or interpreting data related to powerful lower limbs' muscles contractions activities.