Context: Hypoglycemia-associated autonomic failure (HAAF) limits the ability of patients with diabetes to achieve target glycemia. Animal models have provided insights into the pathogenesis of HAAF, but a robust human model of HAAF in which recurrent hypoglycemia impacts the counterregulatory responses to hypoglycemia days later is lacking. Objective: The aim of this study was to determine the impact of two or three episodes of moderate hypoglycemia on counterregulatory responses to subsequent hypoglycemia induced 5 days later. Design and Subjects: Six healthy subjects participated in each of the two study protocols. In both protocol 1 and 2, subjects underwent two 2-hour hypoglycemic clamp studies during the morning and afternoon of day 1. In protocol 2, subjects underwent an additional third hypoglycemic clamp during the morning of day 2. All subjects in both protocols underwent a final hypoglycemic clamp on the morning of day 5. Results: In protocol 1, there were no significant differences in the hypoglycemia-induced hormone response or in symptoms scores between the mornings of days 1 and 5. In protocol 2, hypoglycemiainduced epinephrine (P=.02) and cortisol (P=.04) secretions were significantly lower on day 5 compared with day 1, whereas glucagon (P=.08) and norepinephrine (P=.59) were not different. Also in protocol 2, neurogenic (P=.02) and neuroglycopenic (P=.04) symptoms during hypoglycemia were decreased on day 5 compared with day 1. Conclusion: These results demonstrate that exposure of healthy humans to three 2-hour hypoglycemic episodes over 30 hours leads to significant blunting in counterregulatory and symptom response to subsequent hypoglycemia on day 5.