OBJECTIVE: Alterations in autonomic function are evident in some chronic pain conditions but have not been thoroughly examined in people with osteoarthritis (OA). The study aimed to examine resting autonomic nervous system (ANS) function in people with knee OA, and the response of the autonomic and nociceptive systems to acute stress. METHODS: A preliminary cross-sectional study was undertaken involving people with knee OA ( = 14), fibromyalgia ( = 13), and pain-free controls ( = 15). The sympathetic and parasympathetic components of the ANS were assessed through measures of pre-ejection period (PEP), skin conductance level (SCL), and high frequency heart rate variability (HF HRV). The nociceptive system was assessed through pain ratings associated with a tonic heat pain stimulus. In separate sessions, ANS and heat pain measures were assessed at rest and in response to nociceptive and mental arithmetic stressors. RESULTS: The knee OA group showed reduced HF HRV at rest and reduced modulation in response to stress. Resting PEP and SCL were normal in the knee OA group but PEP modulation was impaired in both chronic pain groups during nociceptive stress. The expected reduction in tonic heat pain ratings in response to stress was lacking in the knee OA and FM groups. CONCLUSION: Preliminary evidence shows impaired parasympathetic nervous system function at rest and in response to nociceptive and mental stress in people with knee OA, with some evidence of altered sympathetic nervous system function. Impaired ANS function could contribute to ongoing pain experienced, and interventions that target ANS function could be beneficial.