Background: Muscle activity and pain development of fibromyalgia (FM) patients in response to mental stress show inconsistent results, when compared to healthy controls (HCs). A possible reason for the inconsistent results is the large variation in stress exposures in different studies. This study compares muscle responses of FM patients and HCs for different modes and levels of imposed stress, to elucidate features in stress exposures that distinguish stress responses of FM patients from HCs. Methods: Upper trapezius (clavicular and acromial fibers), deltoid, and biceps surface electromyographic (sEMG) activity was recorded in FM patients (n=26) and HCs (n=25). Heart rate (HR) was recorded and used as indicator of autonomic activation. Tests included inspiratory breath holding (sympathetic activation procedure), mental stress tests (color-word test and backward counting; 28 min), instructed rest prior to stress test (30 min TV watching), and controlled arm movement. sEMG and HR was also recorded during an unrestrained evening stay at a patient hotel. The 5-min period with lowest trapezius muscle activity was determined. Pain (shoulder/neck, low back pain) and perceived tension were scored on VAS scales at the start and the end of the stress test and at bedtime. Results: Trapezius sEMG responses of FM patients were significantly higher than HCs during sympathetic activation, mental stress, and instructed rest, but similar during arm movement and unrestrained evening activity. HR of FM patients and HCs was similar during mental stress and in the evening, including the 5-min period with lowest trapezius activity. Muscle activity of FM patients during the stress test (with shoulder/neck pain development) and the evening stay (no pain development) was similar. Conclusions: FM patients show elevated muscle activity (in particular trapezius activity) in situations with imposed stress, including sympathetic activation, and putative anticipatory stress. Muscle activity and HR were similar to HCs in instructed arm movement and in a situation approaching low-stress daily living. Pain development of FM patients during the stress test may be due to activation of several stress-associated physiological systems, and not obviously caused by muscle activity in isolation.