The intestinal L-cell is the principal source of glucagon-like peptide-1 (GLP-1), a major determinant of insulin release. As GLP-1 secretion is regulated in a circadian manner in rodents, we investigated whether the activity of the human L-cell is also time-sensitive. Rhythmic fluctuations in the mRNA levels of canonical clock genes were found in the human NCI-H716 L-cell model, which also showed a time-dependent pattern in their response to well-established secretagogues. A diurnal variation in GLP-1 responses to identical meals (850 kcal), served 12 h apart in the normal dark (23:00) and light (11:00) periods, was also observed in male volunteers maintained under standard sleep and light conditions. These findings suggest the existence of a daily pattern of activity in the human L-cell. Moreover, we separately tested the short-term effects of sleep-deprivation and nocturnal light exposure on basal and postprandial GLP-1, insulin and glucose levels in the same volunteers. Sleep-deprivation with nocturnal light exposure disrupted the melatonin and cortisol profiles, and increased insulin resistance. Moreover, it also induced profound derangements in GLP-1 and insulin responses, such that postprandial GLP-1 and insulin levels were markedly elevated and the normal variation in GLP-1 responses was abrogated. These alterations were not observed in sleep-deprived participants maintained under dark conditions, indicating a direct effect of light on the mechanisms that regulate glucose homeostasis. Accordingly, the metabolic abnormalities known to occur in shift-workers may be related to the effects of irregular light-dark cycles on these glucoregulatory pathways.