The current study was designed to investigate how varied time of day (ToD) could affect the acquisition of basketball game actions from different video formats. First-year physical education students (i.e., novice practitioners) were quasi-randomly assigned to three experimental conditions: continuous video, macro-segmented video, or micro-segmented video. Morning (between 08:00 and 09:00) and late afternoon (between 16:00 and 17:00) sessions were conducted, involving study phases (i.e., observation of the scenes of play) and immediate-recall tests (i.e., game comprehension test and game performance test). Oral temperature and mood states were also collected at the beginning of each session. Compared to the morning, the results revealed that afternoon hours resulted in higher oral temperature, higher negative mood states (e.g., anxiety and fatigue), and lower positive mood (i.e., vigor) in all experimental conditions. For both short-term learning tests, the results showed that: (i) students performed significantly better in the morning rather than in the late afternoon (regardless of video formats), (ii) students exposed to the two segmented videos performed better than those exposed to a continuous video at the two ToD (without any significant difference between macro-segmented and micro-segmented videos). The findings highlight the morning's superiority in the acquisition of motor knowledge from different video formats, due to mood disturbances and lower arousal levels. The present experiment encourage basketball teachers to use segmented videos, especially in the morning, to explain tactical information for novices.