The aim of the present study was to investigate reactions in trapezius muscle blood flow (MBF), muscle activity, heart rate variability (HRV) and systemic blood pressure (BP) to autonomic tests in subjects with chronic neck-shoulder pain and healthy controls. Changes in muscle activity and blood flow due to stress and unfavourable muscle loads are known underlying factors of work-related muscle pain. Aberration of the autonomic nervous system (ANS) is considered a possible mechanism. In the present study, participants (n = 23 Pain, n = 22 Control) performed autonomic tests which included a resting condition, static hand grip test (HGT) at 30% of maximal voluntary contraction, a cold pressor test (CPT) and a deep breathing test (DBT). HRV was analysed in time and frequency domains. MBF and muscle activity were recorded from the upper trapezius muscles using photoplethysmography and electromyography (EMG). The pain group showed reduced low frequency-HRV (LF) and SDNN during rest, as well as a blunted BP response and increased LF-HRV during HGT (∆systolic 22 mm Hg; ∆LF(nu) 27%) compared with controls (∆systolic 27; ∆LF(nu) 6%). Locally, the pain group had attenuated trapezius MBF in response to HGT (Pain 122% Control 140%) with elevated trapezius EMG following HGT and during CPT. In conclusion, only HGT showed differences between groups in systemic BP and HRV and alterations in local trapezius MBF and EMG in the pain group. Findings support the hypothesis of ANS involvement at systemic and local levels in chronic neck-shoulder pain.