OBJECTIVE: To examine the association between physical activity measured during leisure, sport, and work and retinal microvascular signs. METHODS: Participants of the Atherosclerosis Risk in Communities (ARIC) Study, a population-based cross-sectional study, had retinal photographs taken at their third follow up visit (1993-1995). Retinal microvascular signs were assessed using a standardized protocol and retinal vascular caliber by a computer-assisted method. Leisure, sport, and work-related physical activity levels were determined through a modified Baecke physical activity questionnaire. RESULTS: A higher level of physical activity during sport and work was significantly associated with a lower prevalence of arteriovenous (AV) nicking, wider venular caliber, and retinopathy. In multivariate models, persons with a level of sport-related physical activity above the median were less likely to have AV nicking (odds ratio [OR] = 0.87; 95% confidence interval [CI] 0.78-0.97) and wider retinal venules (OR = 0.91; 95% CI: 0.83-0.99). Persons with a level of work-related physical activity above the median were less likely to have diabetic retinopathy (OR = 0.66, 95% CI: 0.51-0.85). CONCLUSIONS: In this cross-sectional analyzes, higher levels of physical activity was associated with a lower prevalence of retinal microvascular abnormalities.