Fibromyalgia (FM) is a common chronic primary pain syndrome in which underlying mechanisms remain largely unknown. The role of the autonomic nervous system (ANS) and interoceptive accuracy (IA), the ability to accurately sense internal body signals, in relation to pain sensitivity was examined in persons with FM which were compared to healthy controls. ANS activity was expected to differ in FM compared to controls, both at rest and while performing mental stressful tasks, and IA was expected to be different too. It was further explored whether IA mediates the relationship between ANS activity and pain. In total 108 participants (n=54 healthy controls and n=54 persons with FM) underwent ANS measures during alternating rest and mental stress conditions. These ANS measures consisted of skin conductance levels (SCL), heart rate (HR) and HR variability (HRV), cardiac output, pre-ejection period and stroke volume (SV). IA was measured using a heartbeat perception task and pain sensitivity was assessed by determining pressure pain thresholds. Several questionnaires were administered which were associated to ANS, IA and pain measures. During mental stress as compared to rest conditions, higher levels were found for SCL (p <.001) and HR (p <.001) in both groups, while SV (p <.004) was higher in rest versus in mental stress. Significant interactions between condition (rest versus mental stress) and groups (FM versus controls) were found for SCL (p =.04) and HR (p =.01), pointing to lower reactivity to stress in FM. Posthoc tests showed no significant group effects for the different conditions (i.e., for the rest or stress conditions). Group main effects were neither found for any of the ANS measures. Lower IA scores were found in FM compared to controls (p =.009). IA did not significantly mediate the relationship between ANS measures and pain thresholds. Since no differences between persons with FM and controls were found for any of the ANS measures, it can be concluded that ANS functions do not seem to be a crucial underlying mechanism in FM, at least in the relatively healthy subgroup included in this study. Importantly however, results showed that IA is diminished in FM. The role of IA in the pathogenesis and treatment of (at least a subgroup of persons with) FM requires further study. Also, studies into interoceptive modalities other than heartbeat perception are needed.