BACKGROUND: Identifying periods of the day which are susceptible to varying levels of physical activity (PA) may help identify key times to intervene and potentially change preschool children's PA behaviors. This study assessed variability of objectively measured moderate-to-vigorous physical activity (MVPA) during weekdays and weekend days among preschool children. METHODS: One hundred and eighty-eight children (aged 3 to 5 years; 53.2% boys) from a northwest English city wore uni-axial accelerometers for 7 consecutive days. RESULTS: Higher levels of MVPA were recorded in boys, particularly those who attended preschool for a half day. Children who attended preschool for a full day engaged in 11.1 minutes less MVPA than children who attended for a half day. After-school hours were characterized by a decrease in activity for all groups. Patterns of activity during the weekend were smoother with less variability. CONCLUSION: This study identified discrete segments of the week, specifically afterschool and during the weekend, when preschoolers engage in low levels of PA. Higher levels of MVPA among children who attended preschool for less time each day suggests that the structured preschool environment is related to decreased activity. Consequently, there is a need for interventions in young children to focus on school and home environments.