OBJECTIVE: It is not clear whether physical activity and sedentary behavior affect retinal microvascular caliber. We investigated associations among physical activity (outdoor and indoor sporting activities), sedentary behaviors (including screen time, television [TV] viewing, and computer and videogame usage), and retinal microvascular caliber in schoolchildren. METHODS AND RESULTS: Six-year-old students (1765/2238) from a random cluster sample of 34 Sydney schools were examined. Parents completed questionnaires about physical and sedentary activities. Retinal images were taken, and retinal vessel caliber was quantified. After adjusting for age, sex, ethnicity, eye color, axial length, body mass index, birth weight, and mean arterial blood pressure, children who spent more time in outdoor sporting activities (in the highest tertile of activity) had 2.2 μm (95% CI 0.65 to 3.71) wider mean retinal arteriolar caliber than those in the lowest tertile (Ptrend=0.004). Increasing quartiles of time spent watching TV were associated with narrower mean retinal arteriolar caliber≈2.3 μm (95% CI 0.73 to 3.92), Ptrend=0.003. CONCLUSIONS: These data suggest that physical activity could have a beneficial influence, whereas screen time has a potential adverse influence on retinal microvascular structure. The magnitude of arteriolar narrowing associated with each hour daily of TV viewing is similar to that associated with a 10-mm Hg increase in systolic blood pressure in children.