ObjectiveTo examine device-measured physical activity levels and sedentary behavior participation during different times of the day (ie, morning, midday, and evening) in adults with multiple sclerosis (MS) who differed in fatigue status. DesignCross-sectional survey. SettingRemote survey study managed by a university-based research laboratory. ParticipantsA population-based sample of 1000 participants with MS were sent recruitment materials by the North American Research Committee on Multiple Sclerosis and 218 participants completed all relevant outcomes (N=218). InterventionsNot applicable. Main Outcome MeasuresParticipants (N=218) completed the Fatigue Severity Scale (FSS) as a measure of fatigue severity and were divided into subgroups of fatigued (FSS score ≥4) and nonfatigued (FSS score <4). Participants wore an ActiGraph GT3X+ on the nondominant hip for 7 days to measure physical activity (ie, light physical activity [LPA], moderate-to-vigorous physical activity [MVPA], steps) and sedentary behavior. ResultsFatigued participants engaged in less MVPA (F[1216]=18.5, P<.001), fewer steps (F[1216]=27.8, P<.001), and more sedentary time (F[1216]=8.2, P=.005) than nonfatigued participants. Regardless of group, the highest levels of LPA (F[1.7355.7]=72.9, P<.001) and MVPA (F[1.8395.3]=23.0,P<.001) occurred in the morning and middle of the day, with the lowest levels in the evening. Regardless of group, the highest levels of sedentary behavior occurred in the evening, with similar levels in the morning and evening (F[1.6354.5]=84.3,P<.001). Regardless of group, participants had more steps in the middle of the day, followed by morning and then evening (F[1.8383.9]=84.7,P<.001). ConclusionsOur results suggest that physical activity timing should be considered in future development and delivery of behavior interventions that focus on increasing physical activity and reducing sedentary behavior among adults with MS who have fatigue.