Health behaviors of physical activity and sleep are critical to the prevention of numerous chronic diseases. The health behaviors of healthcare professionals are even more critical, as healthcare providers who practice positive health behaviors are more likely to promote these healthy behaviors in their patients. To assess the health status and health behaviors of medical students, faculty, and staff in an academic health center in the US, and examine the associations between behaviors, physical and mental health outcomes and burnout. Students, faculty, residents and staff from a large university medical system completed an online survey between late-September and mid-November 2019. Associations were examined between health behaviors and health status including mental health outcomes with burnout. Participating in any leisure time physical activity and having a Pittsburgh Sleep Quality Index score < 5 were associated with fewer physical health conditions and lower odds of reporting pain at any site (n = 2060; students n = 242, residents n = 32, staff n = 1425, faculty n = 361). Leisure physical activity and fewer sleep symptoms were associated with fewer reported depressive, anxiety and stress-related symptoms. Participating in leisure physical activity and good-quality sleep were associated with lower odds of burnout. The current study found high rates of physical inactivity and poor sleep among medical students, faculty and staff at an academic health center. These health behaviors were associated with poor mental health and high burnout. Programs and policies are needed improve these health behaviors to reduce burnout. The online version contains supplementary material available at 10.1007/s11332-022-00902-7.