Serious adverse cardiovascular events peak in the morning, possibly related to increased thrombosis in critical vessels. Plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis, is a key circulating pro-thrombotic factor that rises in the morning in humans. We tested whether this morning peak in PAI-1 is caused by the internal circadian system or by behaviors that typically occur in the morning, such as altered posture and physical activity. Twelve healthy adults underwent a 2-week protocol that enabled distinction of endogenous circadian effects from behavioral and environmental effects. The results demonstrated a robust circadian rhythm in circulating PAI-1 with a peak corresponding to ~6:30 AM. This rhythm in PAI-1 was 8-times larger than changes in PAI-1 induced by standardized behavioral stressors including head-up tilt and 15-min cycle exercise. If this large endogenous morning peak in PAI-1 persists in vulnerable individuals, it could help explain the morning peak in adverse cardiovascular events.