Context: Chronobiological factors may modulate the impact of sleep loss on glucose homeostasis. However, these interactions have not been systematically assessed in humans.Objective: To assess the effect of sleep loss during the late vs early night on glucose homeostasis.Design: Fifteen normal-weight men participated in three conditions of a randomized, balanced crossover study comprising two conditions with shortened sleep (i.e., 4 hours of sleep during the first or the second half of the night) and a control condition with 8 hours of sleep. Glucose, insulin, cortisol, and glucagon were measured. Insulin sensitivity and secretion were assessed with a Botnia clamp.Results: Compared with regular sleep duration, sleep loss reduced insulin sensitivity (M-value; P = 0.031) irrespective of early-or late-night timing (P = 0.691). The disposition index (i.e., the beta-cell response adjusted for insulin sensitivity) also tended to be impaired by short sleep (P = 0.056) but not by sleep timing (P = 0.543). In contrast, sleep loss in the second half but not the first half of the night induced reductions in morning glucagon and cortisol levels (P < 0.031) followed by a transient increase in cortisol (P < 0.044).Conclusions: Although sleep deprivation acutely reduced insulin sensitivity irrespective of its nocturnal timing, sleep loss in the early morning compromised a-cell and hypothalamic-pituitary-adrenal axis activity to a greater extent than sleep loss in the first half of the night. This pattern suggests that the timing of sleep restriction can partly potentiate its deleterious metabolic effects.