Chronic musculoskeletal pain (CMP) conditions, like fibromyalgia, are associated with widespread pain and alterations in autonomic functions. Regular physical activity prevents the development of CMP and can reduce autonomic dysfunction. We tested if there were alterations in autonomic function of sedentary mice with CMP, and whether exercise reduced the autonomic dysfunction and pain induced by CMP. Chronic musculoskeletal pain was induced by 2 intramuscular injections of pH 5.0 in combination with a single fatiguing exercise task. A running wheel was placed into cages so that the mouse had free access to it for either 5 days or 8 weeks (exercise groups) and these animals were compared to sedentary mice without running wheels. Autonomic function and nociceptive withdrawal thresholds of the paw and muscle were assessed before and after induction of CMP in exercised and sedentary mice. In sedentary mice, we show decreased baroreflex sensitivity, increased blood pressure variability, decreased heart rate variability, and decreased withdrawal thresholds of the paw and muscle 24 hours after induction of CMP. There were no sex differences after induction of the CMP in any outcome measure. We further show that both 5 days and 8 weeks of physical activity prevent the development of autonomic dysfunction and decreases in withdrawal threshold induced by CMP. Thus, this study uniquely shows the development of autonomic dysfunction in animals with chronic muscle hyperalgesia, which can be prevented with as little as 5 days of physical activity, and suggest that physical activity may prevent the development of pain and autonomic dysfunction in people with CMP.