This study examined the effect of time of day (TOD) on physical performance, and physiological and perceptual responses to a 10-km cycling time trial (TT
10km
). Twelve physically trained subjects (20.3 ± 1.2 years, 74.3 ± 7.4 kg, 179.7 ± 5.5 cm) completed, in a randomized order, a TT
10km
in the morning and in the evening. Intra-aural temperature (IAT) was measured at rest and following the TT
10km
. Completion time, power output (PO), rating of perceived exertion (RPE), heart rate (HR), minute ventilation (V̇E), oxygen uptake (V̇O
2
), carbon dioxide production (V̇CO
2
) and respiratory exchange ratio (RER) were assessed every km during the TT
10km
. Blood lactate concentration [La] and blood glucose concentration [Glu] were assessed before, during and immediately after the TT
10km
. Faster completion time (Δ = 15.0s, p = 0.03) and higher IAT (Δ = 0.33°C, p = 0.02 for pre-TT
10km
) were obtained in the evening compared to the morning with a significant correlation between Δ completion time and Δ IAT at post-TT
10 km
(r = -0.83, p = 0.04). V̇O
2
, [La] and [Glu] increased significantly during both test sessions (p < 0.001) with higher values in the evening compared to the morning (p = 0.015, p = 0.04, p = 0.01, respectively). However, the remaining parameters were found to be only affected by the TT
10km
(p < 0.001). The TT
10km
generates a higher V̇O
2
and higher [La] and [Glu] responses, contributing to a better cycling performance in the evening compared to the morning. The similar magnitude of the TOD effect on completion time and IAT at post-TT
10km
confirms that core temperature is one of the underlying factors contributing to the diurnal variation in physical performance.