Temporal associations between moderate-to-vigorous physical activity (MVPA) at four different multi-hour intervals during the day and the following indicators of sleep quality and sleep quantity in adolescents were examined. This research is a part of the CRO-PALS study, including 129 adolescents (48 boys, 81 girls; mean age ± SD = 15.6 ± 0.4 years) having complete data on sleep and MVPA measured by the SenseWear Pro3 Armband monitor. As data had a hierarchical structure, repeated measures multilevel modelling was used to assess the associations between PA and sleep. During school nights, in girls, morning MVPA was not related to following indicators of sleep quality and sleep quantity. At the same time, evening MVPA was linked with longer sleep onset latency (β = 0.064; 95%CI = 0.025 to 0.103) and higher wake after sleep onset (β = 0.156; 95%CI = 0.0482 to 0.2638). Moreover, during weekend nights, morning MVPA was not associated with sleep, while evening MVPA was accompanied by longer sleep onset latency (β = 0.058; 95%CI = 0.023 to 0.093), higher wake after sleep onset (β = 0.104; 95%CI = 0.012 to 0.20), and negatively with sleep efficiency (β = −0.019; 95%CI = −0.037 to −0.001). In boys the specific multi-hour interval of performing physical activity was not associated with any of indicators of sleep quality. In conclusion, while no strong relations between MVPA and sleep were seen among boys, in girls morning hours MVPA resulted in better sleep patterns over school nights, while afternoon MVPA was followed with slight worsening of some indicators of sleep quality across the whole week. •In boys, the timing of physical activity was not associated with subsequent sleep.•In girls, morning physical activity improved subsequent sleep quality on school nights only.•Evening physical activity impaired sleep quality in girls across the whole week.