Secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1), by the intestinal L-cell is rhythmically regulated by an independent molecular clock. However, the impact of factors known to affect the activity of similar cell-autonomous clocks, such as circulating glucocorticoids and high-fat feeding, on GLP-1 secretory patterns remains to be elucidated. Herein the role of the endogenous corticosterone rhythm on the pattern of GLP-1 and insulin nutrient-induced responses was examined in corticosterone pellet-implanted rats. Moreover, the impact of nutrient excess on the time-dependent secretion of both hormones was assessed in rats fed a high-fat, high-sucrose diet. Finally, the effects of the saturated fatty acid, palmitate, on the L-cell molecular clock and GLP-1 secretion were investigated in vitro using murine GLUTag L-cells. Diurnal variations in GLP-1 and insulin nutrient-induced responses were maintained in animals lacking an endogenous corticosterone rhythm, suggesting that glucocorticoids are not the predominant entrainment factor for L-cell rhythmic activity. In addition to hyperglycemia, hyperinsulinemia, insulin resistance, and disorganization of feeding behavior, high-fat high-sucrose-fed rats showed a total abrogation of the diurnal variation in GLP-1 and insulin nutrient-induced responses, with comparable levels of both hormones at the normal peak (5:00 pm) and trough (5:00 am) of their daily pattern. Finally, palmitate incubation induced profound derangements in the rhythmic expression of circadian oscillators in GLUTag L-cells and severely impaired the secretory activity of these cells. Collectively our findings demonstrate that obesogenic diets disrupt the rhythmic activity of the L-cell, partially through a direct effect of specific nutritional components.