The purpose of this study was to investigate the effects of physical activity and inactivity on the microvasculature in children, as measured from retinal photographs.All participants were from the Hong Kong Children Eye Study, a population-based cross-sectional study of children aged 6 to 8 years. They received comprehensive ophthalmic examinations and retinal photography. Their demographics and involvement in physical activity and inactivity were obtained from validated questionnaires. A validated Deep Learning System was used to measure, from retinal photographs, central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE).In the final analysis of 11,959 participants, 6244 (52.2%) were boys and the mean age was 7.55 (1.05) years. Increased ratio of physical activity to inactivity was associated with wider CRAE (β = 1.033, P = 0.007) and narrower CRVE (β = -2.079, P < 0.001). In the subgroup analysis of boys, increased ratio of physical activity to inactivity was associated with wider CRAE (β = 1.364, P = 0.013) and narrower CRVE (β = -2.563, P = 0.001). The subgroup analysis of girls also showed increased ratio of physical activity to inactivity was associated with narrower CRVE (β = -1.759, P = 0.020), but not CRAE.Increased activity in children is associated with healthier microvasculature, as shown in the retina. Our study contributes to the growing evidence that physical activity positively influences vascular health from a young age. Therefore, this study also underscores the potential of using the retinal vasculature as a biomarker of cardiovascular health.