Key points The growth of new capillaries, angiogenesis, within skeletal muscle occurs only after weeks of repeated aerobic exercise. Paradoxically, large increases in pro‐angiogenic factors such as vascular endothelial growth factor occur with a single exercise bout. The mechanisms underlying the substantial lag in the angiogenic response remain to be elucidated. We detected concomitant increases in the angiostatic Forkhead Box ‘O’ transcription factors FoxO1 and FoxO3a and the matrix protein thrombospondin‐1 following a single bout of exercise, but these responses were repressed after 10 days of repeated exercise. This observation led us to hypothesize that FoxO proteins delay the initiation of exercise‐induced angiogenesis. Endothelial cell‐directed deletion of FoxO proteins abolished the increase in thrombospondin‐1 following a single exercise bout, and resulted in a substantially accelerated angiogenic response. This study identifies an intrinsic endothelial‐specific FoxO signalling pathway that opposes the onset of physiological angiogenesis within healthy exercising skeletal muscle and demonstrates that endothelial cell FoxO proteins are critical determinants of the angiogenic capacity within skeletal muscle. AbstractThe physiological process of exercise‐induced angiogenesis involves the orchestrated upregulation of angiogenic factors together with repression of angiostatic factors. The Forkhead Box ‘O’ (FoxO) transcription factors promote an angiostatic environment in pathological contexts. We hypothesized that endothelial FoxO1 and FoxO3a also play an integral role in restricting the angiogenic response to aerobic exercise training. A single exercise bout significantly increased levels of FoxO1 and FoxO3a mRNA (5.5‐ and 1.7‐fold, respectively) and protein (1.7‐ and 2.2‐fold, respectively) within the muscles of mice 2 h post‐exercise compared to sedentary. Training abolished the exercise‐induced increases in both FoxO1 and FoxO3a mRNA and proteins, and resulted in significantly lower nuclear levels of FoxO1 and FoxO3a protein (0.5‐ and 0.4‐fold, respectively, relative to sedentary). Thrombospondin 1 (THBS1) protein level closely mirrored the expression pattern of FoxO proteins. The 1.7‐fold increase in THBS1 protein following acute exercise no longer occurred after 10 days of repeated exercise. Endothelial cell‐directed conditional deletion of FoxO1/3a/4 in mice prevented the increase in THBS1 mRNA following a single exercise bout. Mice harbouring the endothelial FoxO deletion also demonstrated a significant 20% increase in capillary to muscle fibre ratio after only 7 days of training while 14 days of training was required to elicit a similar increase in wildtype littermates. Our results demonstrate that the downregulation of FoxO1 and FoxO3a proteins facilitates angiogenesis in response to repeated exercise. In conclusion, FoxO proteins can delay exercise‐induced angiogenesis, and thus are critical regulators of the physiological angiogenic response in skeletal muscle.