Hypoglycemia frequently occurs during or following exercise in intensively treated patients with type-1 diabetes mellitus (T1DM), but the underlying mechanisms are not clear. In both diabetic and non-diabetic subjects, moderate hypoglycemia blunts counterregulatory responses to subsequent exercise, but it is unknown whether milder levels of hypoglycemia can exert similar effects in a dose dependent fashion. This study was designed to test the hypothesis that prior hypoglycemia of differing depths induces acute counterregulatory failure of proportionally greater magnitude during subsequent exercise in T1DM. Twenty-two T1DM patients (11m / 11f, HA1c 8.0±0.3), were studied during 90 min of euglycemic cycling exercise, following two 2-hr periods of previous day euglycemia (EUGLY) or hypoglycemia of 3.9, 3.3 or 2.8 mmol/L (HYPO- 3.9, HYPO-3.3, HYPO-2.8, respectively). Patients' counterregulatory responses (circulating levels of neuroendocrine hormones, intermediary metabolites, substrate flux, tracer determined glucose kinetics and cardiovascular measurements) were assessed during exercise. Identical euglycemia and basal insulin levels were successfully maintained during all exercise studies, regardless of blood glucose levels during the previous day. Following day 1 euglycemia, patients displayed normal counterregulatory responses to exercise. Conversely, when identical exercise was performed following day 1 hypoglycemia of increasing depth, a progressively greater blunting of glucagon, catecholamine, cortisol, endogenous glucose production and lipolytic responses to exercise were observed. This was paralleled by a graduated increase in the amount of exogenous glucose needed to maintain euglycemia during exercise. Our results demonstrate that acute counterregulatory failure during prolonged, moderate-intensity exercise may be induced in a dose dependent fashion by differing depths of antecedent hypoglycemia starting at only 3.9 mmol/L in patients with T1DM.