Abstract: Studies investigating the relationship between physical activity and sleep have mostly been cross-sectional, inconclusive, and use subjective means of physical activity and sleep data. This longitudinal prospective study, using a hip accelerometer, investigates how increasing daytime moderate-vigorous physical activity along with a decrease in sedentary behavior affect children's sleep. Methods: In the first study, the hip worn accelerometer was validated to the wrist worn accelerometer in measuring sleep. In a second study, fifth grade children (n=100) participating in after school activity programs located in San Francisco, CA wore an accelerometer on the hip for 3 weekdays and parents filled out a parental assessment of child's sleep. Bedtime and final wake time were ascertained by researcher estimate. Total sleep time (TST) was calculated from each unit using Sadeh's scoring routine for children's sleep. Primary analysis used Pearson's correlations for associations between the physical activity measures and TST. The association between obesity and the physical activity/sleep relationship was explored using multivariate linear regression. Results: Mean weekday MVPA significantly increased by 1.1% (2.3%) (p=0.0001), mean weekday sedentary activity significantly decreased by 2.6% (6.0%) (p=0.0001) and mean weekday TST decreased by 16 min (43.3) (p=0.0006). Correlations for the association of the difference in MVPA and difference in sedentary activity with the difference in TST were r = 0.01 and r = -0.05. Discussion: No association was found between increased MVPA and decreased sedentary activity and difference in TST in both the overweight and non-overweight. Further longitudinal studies, with larger increases in MVPA, are needed to investigate the relationship of MVPA and sleep duration and behavior.