Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays a central role in neuronal health. BDNF exists in two primary isoforms, the mature form (mBDNF) and its precursor (proBDNF), with opposing downstream effects on neuronal function. The positive effect of exercise on plasma levels of the BDNF isoforms has been extensively studied in adults. However, equivalent investigations are lacking in children and adolescents. Twenty healthy children (9-12 years old), 19 adolescents (13-17 years old) and 39 adults (23-49 years old) donated venous blood before and after a 45-minute run. Platelet-poor plasma was analysed for pro- and mBDNF using an enzyme-linked immunosorbent assay. Maximal oxygen uptake and anthropometric data were assessed in all participants, while Tanner stage, circulating sex hormones and accelerometry-based activity level were assessed in children and adolescents only. We found that children, adolescents and adults have similar circulating levels of plasma pro- and mBDNF at rest. For children and adolescents, resting levels of mBDNF correlated with average time spent in vigorous activity. In response to the acute endurance exercise intervention, mBDNF increased in all age groups, but the greatest rise in mBDNF was seen in adults. The acute endurance exercise did not affect proBDNF levels. Our results demonstrate that plasma mBDNF levels, but not proBDNF, increase following endurance exercise in all age groups, with a greater effect in adults. We also show that high-intensity physical activity, but not underlying fitness, is contributing to sustained elevated mBDNF levels. KEY POINTS: We show that in children and adolescents, regular vigorous physical activity is key to increased basal levels of plasma mature brain-derived neurotrophic factor (mBDNF), a factor linked to neuroplasticity and brain health. The ability to elevate mBDNF through exercise is present across all age groups, with the greatest increase in adults. The mBDNF response to physical exercise seems to be independent of underlying physical fitness. Our findings suggest that basal plasma mBDNF levels may reflect the cumulative effects of repeated exercise rather than an individual's overall physical fitness.