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Background The prevention and rehabilitation of multisite musculoskeletal pain would benefit from studies aiming to understand its underlying mechanism. Autonomic imbalance is a suggested mechanism for multisite pain, but hardly been studied during normal daily living. Therefore, the aim of the study is to investigate the association between multisite musculoskeletal pain and cardiac autonomic modulation during work, leisure and sleep. Methods This study is based on data from the “Danish Physical activity cohort with objective measurements” among 568 blue-collar workers. Pain intensity scales were dichotomized according to the median of each scale, and the number of pain sites was calculated. No site was regarded as the pain-free, one site was considered as single-site musculoskeletal pain and pain in two or more sites was regarded as multisite musculoskeletal pain. Heart rate variability (HRV) was measured by an electrocardiogram system (ActiHeart) and physical activity using accelerometers (Actigraph). Crude and adjusted linear mixed models were applied to investigate the association between groups and cardiac autonomic regulation during work, leisure and sleep. Results There was no significant difference between groups and no significant interaction between groups and domains in the crude or adjusted models for any HRV index. Significant differences between domains were found in the crude and adjusted model for all indices, except SDNN; sleep time showed higher values than leisure and work time, except for LF and LF/HF, which were higher during work. Conclusion This cross-sectional study showed that multisite musculoskeletal pain is not associated with imbalanced cardiac autonomic regulation during work, leisure and sleep time. Electronic supplementary material The online version of this article (10.1186/s12891-018-2312-3) contains supplementary material, which is available to authorized users.