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Shift work is increasingly being suggested to be associated with an increased risk for overweight. Physical activity (PA) has been hypothesised to play a role in the health effects of shift work, but research on this role of PA is scarce. This study aimed to examine the association between night shift work and body mass index (BMI) and the moderating role of PA therein.Data from 588 workers were used from the prospective cohort Klokwerk+ study, examining the health effects of night shift work in health care workers. BMI was calculated by measured body weight (in kg) divided by body height (in metre squared). PA was measured using the validated Short QUestionnaire to ASses Health-enhancing PA (SQUASH) questionnaire. Linear regression analyses were performed for the associations between shift work and BMI; interaction terms were added to determine the influence of PA.Mean BMI for shift workers was 25.3 (SD=4.2) versus 25.4 (SD=4.2) for non-shift workers (p>0.05). Shift workers were more moderately active than non-shift workers (beta 318 min/wk, 95% CI 141–496). After adjustment, there were no significant differences in the amount of vigorous intensity PA (beta-43 min/wk, 95% CI-115-26). There was no significant interaction for either moderate or vigorous PA in the shift work-BMI association.Our study could not confirm the hypothesis that PA moderates the shift work-BMI relation. To confirm these findings and to get more insight into the moderating and mediating role of PA and other lifestyle behaviours, more longitudinal studies are recommended.