We have evaluated the influence of fasting on the response of counterregulatory hormones to insulin-induced hypoglycaemia. Eight healthy, non-obese volunteers were studied after an overnight fast and again after a 72-h fast period. Levels of blood glucose were higher after overnight fasting (4.59±0.10 mmol/1) than after 72 h of fasting (3.38±0.12 mmol/1). Hypoglycaemia was induced by a constant insulin infusion (2.4 mU/kg/min) and clamped between 2.1 and 2.3 mmol/1 of glucose by a variable glucose infusion. Hypoglycaemia evoked stimulation of glucagon release after the overnight fast but did not alter release after 72 h of fasting. The response of other counterregulatory hormones were also influenced by the longer fasting period: the normal rise in adrenaline levels during hypoglycaemia was delayed and attenuated and the normal rise in Cortisol levels was absent; paradoxically, Cortisol levels decreased during hypoglycaemia. Seventy-two hours of fasting, therefore, profoundly alters hormonal responses to hypoglycaemia.