This study sought to examine how the physical activity levels and sedentary time of preschoolers attending center-based childcare varied across the week. Sex differences were also explored. Participants (n=101) wore Actical™ accelerometers (15 s epoch) for five consecutive days during childcare hours only. A multivariate repeated measures analysis of variance was used to evaluate levels of sedentary, light, and moderate-to-vigorous (MVPA) physical activity across the five weekdays. Total physical activity (TPA) was analyzed separately in a univariate repeated measures ANOVA. Sex was entered as an additional between-subjects factor. Levels of sedentary time, LPA, and TPA across the week were found to be statistically significant, and can best be described by quadratic effects. Participants' activity levels and sedentary time typically peaked mid-week. Levels of physical activity and sedentary time were not found to significantly differ based on sex. Childcare centers may benefit from the introduction and/or modification of active play-based programming and curricula, particularly at the start and end of the week where preschoolers' activity levels tend to be lower. Additional investigations are required to confirm these findings.