Background and aims Physical activity (PA) and fitness are important modulators of vascular ageing and may therefore help expand individual health span. We aimed to systematically review the association of PA and fitness, as well as the effects of exercise interventions on the new microvascular biomarkers retinal arteriolar (CRAE) and venular (CRVE) diameters and the retinal flicker light-induced dilatation (FID) in children and adults. Methods PubMed, Ovid, The Cochrane, EMBASE and Web of Science were searched. 805 studies were found, and 25 full-text articles analysed. Twenty-one articles were included in this systematic review. Results Higher PA levels were associated with narrower CRVE in children and adults. Physical inactivity was associated with wider CRVE in both age groups. Combined aerobic and motor skill training in school settings lead to wider CRAE in children. Aerobic exercise interventions in adults with or without CV risk factors induced wider CRAE and narrower CRVE. Studies on the effect of exercise on FID are scarce. In a twelve-week randomized controlled trial, high-intensity interval training significantly improved FID in older patients with CV risk factors. Conclusions Higher PA and fitness levels were associated with improved retinal microvascular health in children and adults. Short-term exercise interventions in healthy children and adults, as well as CV risk patients, improved retinal microvascular structure and function. Exercise has the potential to counteract microvascular remodelling and development of small vessel disease during lifespan. Retinal vessel analysis can differentiate the beneficial effects of exercise on target microvascular organ damage.