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Shift work may alter workers’ physical activity (PA) level, making PA a potential underlying mechanism of the negative health effects of shift work. As prior studies on shift work and PA have generally used self-reported, overall PA measures, the results may be susceptible to bias. Therefore, our aim was to compare objectively measured non-occupational and occupational PA levels between shift workers and non-shift workers.Data were used from Klokwerk+, a prospective cohort study examining the health effects of shift work among health care workers. In total, 401 rotating and/or night shift workers and 78 non-shift workers were included, who wore Actigraph GT3X+ accelerometers for 7 consecutive days. Time spent sitting, standing, walking, running, stairclimbing, and cycling during leisure and at work was estimated using Acti4-software. Linear regression was used to compare proportions of time spent in these activities between shift and non-shift workers.Average accelerometer wear-time was 105.9 hours (SD=14.0) over an average of 6.9 days (SD=0.6). No differences between shift workers and non-shift workers were found in PA behaviours during leisure-time (p>0.05). At work, shift workers were less sedentary (B=−10.6 (95%-CI=−14.3- −6.8)) and spent larger proportions of the time standing (B=9.5 (95%-CI=6.4–12.6)) and walking (B=1.2 (95%-CI=0.1–2.2)) than non-shift workers.Non-occupational PA levels of shift workers were similar to that of non-shift workers, but shift workers were more physically active (i.e. standing/walking) at work. Future research should focus on the role of this difference in occupational PA in the health effects of shift work.