Objective To test the hypotheses that dysregulation of the autonomic nervous system (ANS) is associated with the presence of chronic widespread pain (CWP), and that dysregulation of the ANS is associated with higher pain intensity in CWP. Methods Cross-sectional data were obtained from 1,574 subjects (healthy controls as well as persons with depressive and anxiety disorders) participating in The Netherlands Study of Depression and Anxiety. The Chronic Pain Grade was used to assess pain intensity and pain-related disability. Heart rate (HR), SD of the normal-to-normal interval (SDNN), the preejection period (PEP), and respiratory sinus arrhythmia (RSA) were used to assess the ANS. Logistic regression analyses and linear regression analyses were conducted with adjustment for potential confounders. Results No differences in HR, PEP, SDNN, or RSA values were found between CWP subjects and controls after adjustment for confounders. However, lower SDNN and lower RSA were associated with higher pain intensity in subjects with CWP. Conclusion Lower parasympathetic activity, as assessed with SDNN and RSA, is associated with higher pain intensity in subjects with CWP. This large and well-controlled study does not provide evidence for an association between dysregulation of the ANS and the presence of CWP.