There is general agreement that prior hypoglycemia blunts subsequent hypoglycemic counterregulatory responses. However, there is considerable debate concerning the timing and number of prior hypoglycemic episodes required to cause this blunting effect. The aim of this study was to determine whether one episode of hypoglycemia could modify neuroendocrine, metabolic, and symptom responses to hypoglycemia induced 2 h later. A total of 24 (12 male and 12 female) young, healthy, overnight-fasted subjects participated in a series of glucose clamp studies. A total of 16 individuals underwent 2 randomized studies of either identical 2-h morning and afternoon hyperinsulinemic (490 +/- 60 pmol/L) hypoglycemia (2.9 +/- 0.1 mmol/L) separated by 2 h or, at least 2 months later, 2-h morning and afternoon hyperinsulinemic (492 +/- 45 pmol/L) euglycemia (5.1 +/- 0.1 mmol/L). A total of 8 other subjects participated in a single experiment that consisted of 2-h morning hyperinsulinemic (516 +/- 60 pmol/L) euglycemia (5.1 +/- 0.1 mmol/L) and 2-h afternoon hyperinsulinemic (528 +/- 66 pmol/L) hypoglycemia (2.9 +/- 0.1 mmol/L) also separated by 2 h. Morning hypoglycemia significantly (P < 0.01) reduced (33-55%) the responses of epinephrine, norepinephrine, glucagon, GH, cortisol, and pancreatic polypeptide during afternoon hypoglycemia. Hypoglycemic symptoms (primarily neuroglycopenic) were also significantly (P < 0.01) reduced during afternoon hypoglycemia. Plasma glucose, insulin, nonesterified fatty acids, glycerol, lactate, beta-hydroxybutyrate (P < 0.01), GH, and cortisol (P < 0.05) levels were significantly increased at the start of afternoon hypoglycemia following morning hypoglycemia. Morning hypoglycemia created an insulin-resistant state during afternoon hypoglycemia. Despite blunted neuroendocrine responses, glucose infusion rates required to maintain hypoglycemia and increases in glucose oxidation were significantly attenuated during afternoon compared with morning hypoglycemia. This was in marked contrast to euglycemic control experiments where glucose infusion rates and nonoxidative glucose disposal were significantly increased during afternoon relative to morning studies. We conclude that in normal man one episode of prolonged, moderate, morning hypoglycemia can produce substantial blunting of neuroendocrine and symptomatic responses to subsequent near-term hypoglycemia, and the induction of posthypoglycemic insulin resistance can compensate for blunted neuroendocrine responses by limiting glucose flux and specifically glucose oxidation during subsequent near-term hypoglycemia.