Background: Hypoglycaemia-associated autonomic failure (HAAF) with impaired neurogenic and neuroglycopaenic responses occurs in adults following recent, repeated hypoglycaemia. Objective and hypotheses: We aimed to evaluate, whether HAAF also occurs in infants with congenital hyperinsulinism (CHI). Methods: The autonomic, neuroglycopaenic, and glucose counter-regulatory hormonal responses were assessed in a) seven infants with CHI during a controlled fast following initial symptomatic hypoglycaemia and three recent episodes of spontaneous recurrent hypoglycaemia each lasting less than 5 minutes; and in b) seven infants with ketotic hypoglycaemia for control. Results: When repeated hypoglycaemia was provoked, all CHI patients exhibited a complete loss of hypoglycaemic symptoms and signs, along with a global blunting of the counter-regulatory cortisol, glucagon, growth hormone, epinephrine and nor-epinephrine response (median values 256 nmol/L; 23 pmol/L; 5.6 mU/L; 390 pmol/L; and 2.9 nmol/L, respectively). In the controls, hypoglycaemia was always symptomatic with normal counter-regulatory cortisol, glucagon, epinephrine and nor-epinephrine responses (530 nmol/L; 60 pmol/L; 920 pmol/L; and 4.0 nmol/L, respectively). Conclusions: Recurrent hyperinsulinaemic hypoglycaemia even of short duration blunts the autonomic, neuroglycopaenic, and glucose counterregulatory hormonal responses in infants with CHI. Tight, or continuous, glucose monitoring is therefore recommended, especially in conservatively treated patients.