This study was aimed (1) to compare the level of physical activity (PA) between working and nonworking hours and (2) to compare the level of PA during working hours of nurse clinical practitioners (NCPs) with that of nurse managers (NMs). This cross-sectional survey was conducted at a Thai university hospital from October 2015 to March 2016. All randomly selected participants wore an activity tracker on their hip for 5 days, except during bathing and sleeping periods, to record step counts and time points. Of 884 nurses, 289 (142 NCPs and 147 NMs) were randomly selected. The average age was 35.87 years. They spent 9.76 and 6.01 hours on work and nonwork activities, respectively. Daily steps per hour were significantly lower during work than nonwork periods (P < .001). An NCP had significantly higher overall hourly PA (P = .002). The number of steps per hour during work period of NCP was significantly higher than that of NM even after adjusting for age, work experience, and body mass index (P = .034). NCP had higher overall PA than NM, which was partly contributed by work-related PA. Level of PA for a professional with variation of actual work hours should be measured on hourly basis.