We tested the hypothesis that increased endogenous cortisol secretion reduces autonomic neuroendocrine and neurogenic symptom responses to subsequent hypoglycemia. Twelve healthy young adults were studied on two separate occasions, once after infusions of a pharmacological dose of α-(1–24)-ACTH (100 μg/h) from 0930 to 1200 and 1330 to 1600, which raised plasma cortisol levels to ∼45 μg/dl onday 1, and once after saline infusions on day 1. Hyperinsulinemic (2.0 mU · kg⁻¹ · min⁻¹) stepped hypoglycemic clamps (90, 75, 65, 55, and 45 mg/dl glucose steps) were performed on the morning of day 2 on both occasions. These markedly elevated antecedent endogenous cortisol levels reduced the adrenomedullary (P = 0.004, final plasma epinephrine levels of 489 ± 64 vs. 816 ± 113 pg/ml), sympathetic neural (P = 0.0022, final plasma norepinephrine levels of 244 ± 15 vs. 342 ± 22 pg/ml), parasympathetic neural (P = 0.0434, final plasma pancreatic polypeptide levels of 312 ± 37 vs. 424 ± 56 pg/ml), and neurogenic (autonomic) symptom (P = 0.0097, final symptom score of 7.1 ± 1.5 vs. 10.6 ± 1.6) responses to subsequent hypoglycemia. Growth hormone, but not glucagon or cortisol, responses were also reduced. The findings that increased endogenous cortisol secretion reduces autonomic neuroendocrine and neurogenic symptom responses to subsequent hypoglycemia are potentially relevant to cortisol mediation of hypoglycemia-associated autonomic failure, and thus a vicious cycle of recurrent iatrogenic hypoglycemia, in people with diabetes mellitus.