The study investigated the possible association between nocturnal trapezius muscle activity and shoulder and neck pain. Sixty female subjects participated in the study, 33 were classified as pain-afflicted on the basis of shoulder and neck pain reports for the previous 6 months. Electromyographic (EMG) activity was monitored bilaterally from the trapezius in all recordings. EMG recording of the deltoid, biceps, and hand flexors was included for 26 subjects (17 with pain) to provide a comparative basis for evaluation of the trapezius recordings. There was considerable variation in the amount of muscle activity between subjects; however, some subjects presented a continuous, low-level activity pattern throughout the presumed sleep period. Subjects classified as pain-afflicted had significantly higher activity level and longer duration of trapezius EMG activity than the pain-free subjects. The deltoid had significantly more activity than the trapezius, while the biceps and the hand flexors presented similar activity level and duration as the trapezius. Nocturnal trapezius activity was not associated with pain exacerbation the same night. We suggest nocturnal trapezius muscle activity is a pointer to physiological mechanisms that contribute to some forms of shoulder and neck pain, such as trapezius myalgia, but nocturnal muscle activity may not be casually implicated in the pain-induction process.