It is unknown whether a school-based prevention program has the potential to improve microvascular health in children. This study investigates the impact of the school-based lifestyle intervention program JuvenTUM 3 on physical activity, physical fitness, serum biomarkers and microvascular function. We studied 434 children (10-11 years) in a cluster-randomized setting (8 intervention schools, IG; 7 control schools, CG) over 18 months. The school-based prevention program included weekly lifestyle lessons for children with the aim to increase physical activity in and outside of school, physical fitness as well as health behavior. Anthropometric measurements and blood sampling were conducted using standard protocols, physical activity by use of a questionnaire and physical fitness by a 6-item-test battery. Central retinal arteriolar (CRAE) and venular (CRVE) vessel diameters as early marker of vascular dysfunction, as well as the arteriolar-to-venular diameter ratio (AVR), were investigated with a non-mydriatic vessel analyser. School-based physical activity increased in 41% of children in IG (19% in CG, p = 0.038). Improvements in vascular parameters were observed for AVR (increase in 83% of children in IG versus 50% in CG; p < 0.001) and for CRVE (43% of children with retinal venular widening in IG versus 58% in CG, p = 0.019). These vascular improvements were also seen in overweight children for CRAE (p = 0.021) and AVR (p < 0.001). The school-based prevention program JuvenTUM 3 increased physical activity at school inducing favourable effects on retinal microvasculature function. These findings underline the importance of early lifestyle interventions in children for primary prevention of cardiovascular disease. Copyright © 2018 Elsevier B.V. All rights reserved.