Hypoglycemia-associated autonomic failure (HAAF)-reduced autonomic (including adrenomedullary epinephrine) and symptomatic responses to hypoglycemia caused by recent antecedent hypoglycemia-plays a key role in the pathogenesis of defective glucose counter-regulation and hypoglycemia unawareness and thus iatrogenic hypoglycemia in type 1 diabetes. On the basis of the findings that cortisol infusion mimics and deficient or inhibited cortisol secretion minimizes this phenomenon, it has been suggested that the cortisol response to antecedent hypoglycemia mediates HAAF. We tested the hypothesis that any stimulus that releases cortisol, such as exercise, reduces autonomic and symptomatic responses to subsequent hypoglycemia. Thirteen healthy young adults (four women) were studied on three occasions in random sequence: 1) cycle exercise (∼70% peak oxygen consumption) from 0830 to 0930 h and from 1200 to 1300 h on day 1 and hyperinsulinemic (2.0 mU · kg-1 · min-1) stepped hypoglycemic (85, 75, 65, 55, and 45 mg/dl) clamps on day 2, 2) rest on day 1 and identical hypoglycemic clamps on day 2, and 3) hyperinsulinemic-euglycemic clamps. Exercise raised plasma cortisol concentrations to 16.9 ± 1.9 (0930 h) and 16.6 ± 1.6 μg/dl (1300 h) on day 1. Compared with rest on day 1, exercise on day 1 was associated with reduced epinephrine (P = 0.0113) responses-but not norepinephrine (P = 0.6270), neurogenic symptom (P = 0.6470), pancreatic polypeptide (P = 0.0629), or glucagon (P = 0.0436, but higher) responses-to hypoglycemia on day 2. However, the effect was small. (The final day 2 hypoglycemia epinephrine values were 765 ± 106 pg/ml after rest on day 1 and 550 ± 94 pg/ml after exercise on day 1 compared with 30 ± 6 pg/ml during euglycemia.) These data are consistent with the hypothesis that the cortisol response to hypoglycemia mediates in part the reduced epinephrine response to subsequent hypoglycemia, one key component of HAAF in type 1 diabetes. However, the small effect suggests that an additional factor or factors may well be involved. These data do not support the hypothesis that the cortisol response to hypoglycemia mediates the reduced neurogenic symptom response to subsequent hypoglycemia, another key component of HAAF in type 1 diabetes.