The Effect of physiological changes in nighttime cortisol and glucagon on endogenous glucose production (EGP) and nocturnal glycemia are unknown.To determine the effects of changes in cortisol and glucagon on EGP during the night.Two overnight protocols were conducted. In Protocol 1, endogenous cortisol was blocked with metyrapone and hydrocortisone infused either at constant (constant) or increasing (variable) rates to mimic basal or physiological nocturnal cortisol concentrations. In Protocol 2, endogenous glucagon was blocked with somatostatin and exogenous glucagon was infused at either 'basal' or 'elevated' rates to mimic nocturnal glucagon concentrations observed in ND and T2D individuals. EGP was measured using [3-3H] glucose and gluconeogenesis estimated with 2H2O in all studies.Mayo Clinic Clinical Research Trials Unit, Rochester, MN, USA.In Protocol 1, 34 subjects [17 non-diabetic (ND) and 17 T2D] and in Protocol 2, 39 subjects [21 ND and 18 T2D] were studied.EGP.EGP, gluconeogenesis and glycogenolysis were higher with variable than constant cortisol at 7 AM in T2D subjects. In contrast, nocturnal EGP did not differ in ND subjects between variable vs. constant cortisol. While elevated glucagon increased EGP, glycogenolysis and gluconeogenesis in ND, the data in T2D subjects indicated that EGP and gluconeogenesis, but not glycogenolysis were higher during the early part of the night.Nocturnal hyperglucagonemia but not physiological rise in cortisol, contributes to nocturnal hyperglycemia in T2D due to increased gluconeogenesis.