To define the chronobiology of glucose tolerance and insulin secretion in obesity, nine obese men and nine lean men were studied during constant glucose infusion for 53 h, including 8 h of nocturnal sleep, 28 h of continuous wakefulness, and 8 h of daytime sleep. Blood samples were collected at 20-min intervals to assay glucose, insulin, C-peptide, cortisol, and GH. Sleep was polygraphically monitored. Abnormal temporal profiles of glucose regulation were observed during wakefulness and sleep in obese subjects. During daytime hours, the normal profile of glucose tolerance was reversed, as an improvement, rather than a deterioration, was observed from morning to late evening. This reversal of the daytime pattern appeared to be caused by a dual defect in glucose regulation during the previous night. Indeed, during early sleep, GH secretion was markedly reduced, and the nocturnal rises of glucose and insulin secretion were dampened. During late sleep, obese subjects failed to suppress insulin secretion and plasma glucose, resulting in high morning levels. Comparisons of metabolic and hormonal patterns during nocturnal and daytime sleep suggest that the failure to suppress insulin secretion in late sleep may reflect a relative insensitivity of the beta-cell to acute inhibitory effects of cortisol in addition to insulin resistance.