The incidence of coronary events appears to increase after the menopause and is reported to occur more frequently in the morning hours. Endothelial function has been shown to exhibit diurnal rhythmicity and is attenuated in postmenopausal women. The protocols used to assess this previously have been confounded by environmental factors (e.g. posture, meals, lighting, sleep). Hence uncertainty remains as to whether the reported rhythm in endothelial function is truly circadian and if it changes after the menopause. The effects of time of day, age, menopausal status and dietary fat on brachial artery endothelial (%FMD) and smooth muscle function (%GTN) was investigated in women using non-invasive ultrasound technology. Endothelial and smooth muscle function was significantly reduced in postmenopausal (n = 10) compared to pre-menopausal women (n = 11), with no discernable rhythm in %FMD either under constant routine (CR) or normal study conditions. In contrast, a day-night variation in %FMD was observed in middle-aged pre-menopausal women (n = 11) in CR conditions and in young women (n = 7) under normal study conditions. This variation, however, did not persist when the data were corrected for circadian phase. The melatonin rhythm was also advanced in postmenopausal women in CR conditions, with a significantly earlier acrophase and earlier melatonin onset (P = 0.057; not quite significant). Extended wakefulness during the CR resulted in a similar reduction in alertness and performance in both the pre-menopausal and postmenopausal women. A high fat meal, consumed in the evening, did not affect %FMD in young women although increased TAG levels were observed. The results suggest that the time of day variation in %FMD is not an endogenous circadian rhythm and is rather a result of exogenous factors. Impairment of both %FMD and %GTN with a loss of rhythmicity in endothelial function occurs after the menopause.