Objectives: To investigate whether a higher proportion of night shifts is associated with a higher occurrence of musculoskeletal pain and headaches. Furthermore, to investigate whether sleep duration can mediate this potential association. Method: The study included 684 nurses in rotating shift work (day, evening, night) who responded to a daily questionnaire about working hours, sleep, and pain for 28 consecutive days. The data were treated as cross-sectional data. Results: A negative binomial regression analysis adjusted for age and BMI revealed that working a higher proportion of night shifts is not associated with a higher occurrence of musculoskeletal pain and headaches. On the contrary, those working ≥50 % night shifts had a significantly lower occurrence of pain in the lower extremities than those who worked <25 % night shifts (IRR 0.69 95 % CI 0.51, 0.94). There was no indication of a mediation effect with total sleep time (TST). Conclusion: The results of this study indicate that working a higher monthly proportion of night shifts is not associated with a higher occurrence of musculoskeletal pain and headaches.