The aim of the study was to investigate autonomic nervous system regulation, physical activity (PA) and perceived stress and energy during daily activities in subjects with chronic muscle pain in the neck-shoulders (trapezius myalgia) (n = 23) and symptom-free controls (n = 22). Subjects underwent 24-h objective ambulatory monitoring of heart rate variability (HRV) and PA, and reported their perceived stress and energy in a diary. Standard HRV measures were extracted in time and frequency domains. The volume and pattern of different types of activities were quantified in terms of intensity and duration of walking, and time spent sitting, standing and lying during the 24-h measurement. Results showed shortened inter beat-intervals (higher heart rate) and reduced HRV in the pain group, most pronounced during sleep (p < 0.05). For overall PA, the pain group showed increased lying time, compared to controls (p < 0.05). A different activity pattern was found in the pain group, with reduced leisure time PA and increased PA during morning hours, in comparison with controls (p < 0.05). Both groups demonstrated low levels of perceived stress, whereas reduced energy was observed in the pain group (p < 0.05). In conclusion, monitoring of 24-h HRV demonstrated diminished HRV among persons with chronic neck-shoulder pain. This reflected aberration in autonomic regulation, suggesting reduced parasympathetic activation and increased sympathetic tone as an element in maintenance of chronic muscle pain.