To test the hypothesis that hypoglycemia itself causes reduced neuroendocrine and symptomatic responses to subsequent hypoglycemia, we measured those responses during clamped hypoglycemia (2.8 mM) on consecutive mornings on two occasions, with interval afternoon (1400–1600) hypoglycemia (3 mM) on one occasion and interval afternoon euglycemia (5 mM) on the other, in nine nondiabetic humans. None of the measured responses were reduced by interval euglycemia. In contrast, plasma epinephrine (P < 0.005), glucagon (P < 0.005), pancreatic polypeptide (P < 0.01), cortisol (P < 0.02), and total (P < 0.001), neurogenic (P < 0.001) and neuroglycopenic (P < 0.05) symptom responses to morning hypoglycemia were reduced after interval afternoon hypoglycemia. Thus, a single episode of hypoglycemia caused a generalized reduction of the neuroendocrine and symptomatic responses to subsequent hypoglycemia, a finding that may be important to the pathogenesis of iatrogenic hypoglycemia in insulin-dependent diabetes mellitus.