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High reactivity to psychosocial stress during childhood increases the risk of future psychological and physiological disorders. The main objective of this study was to investigate associations between children's moderate-to-vigorous physical activity (MVPA) and physiological and psychological reactions to acute psychosocial stress. Randomized cross-over study comparing two groups. After a 7-day accelerometer-based measurement of MVPA, 110 children aged 10–13 years (48 female) were categorized into a low (< 30 min/d) or a high MVPA group (> 60 min/d). On separate laboratory appointments, participants completed the Trier Social Stress Test for Children (TSST-C) and a non-stressful control task in randomized order. Stress reactivity was assessed from saliva cortisol, heart rate variability and self-reported anxiety. In repeated measures analyses of variance, main effects of condition indicated higher stress reactivity in the TSST-C compared to control for cortisol and anxiety, with more pronounced effects in girls compared to boys. Differences in heart rate variability disappeared after controlling for confounding variables. No interactions with MVPA were found. A main effect of MVPA indicated generally lower cortisol reactivity in the high compared to the low MVPA group. The TSST-C is suitable for induction of psychosocial stress in the laboratory, but sex-specific differences have to be considered. Children who met the WHO recommendations for MVPA exhibited generally lower endocrine reactivity to both laboratory tasks combined, underlining the potential role of regular physical activity in the regulation of the stress response in early developmental stages. Further studies are necessary to ascertain causal associations. [ABSTRACT FROM AUTHOR]