OBJECTIVE A meal rich in protein stimulates insulin secretion. Long-term effects of dietary protein on insulin release and glucose metabolism are, however, still not known. Our study focussed on the effect of different protein intake on pancreatic insulin secretion capacity, glycogen turnover and gluconeogenesis. METHODS Subjects with constant (6 months) dietary protein of 1.87 +/- 0.26 g kg(-1) day(-1) (1.25-2.41) named high protein group and with 0.74 +/- 0.08 (0.57-0.80), normal protein group, were identified by a food questionnaire and were matched (n = 9) according to sex, age and calorie intake. They underwent an intravenous glucose tolerance test and a euglycaemic hyperinsulinaemic clamp with infusion of [6,6-2H2]-glucose combined with indirect calorimetry. To estimate net gluconeogenesis the usual diet was enriched by deuterated water or U-[13C6]-glucose and breath and plasma were sampled. RESULTS Glucose-stimulated insulin secretion was increased in the high protein group (516 +/- 45 pmol/l vs 305 +/- 32, p = 0.012) due to reduced glucose threshold of the endocrine beta cells (4.2 +/- 0.5 mmol/l vs 4.9 +/- 0.3, p = 0.031). Endogeneous glucose output was increased by 12% (p = 0.009) at 40 pmol/l plasma insulin in the high protein group, but not at higher insulin concentration whereas overall glucose disposal was reduced. Fasting plasma glucagon was 34% increased in the high protein group (p = 0.038). Fractional gluconeogenesis was increased by 40% in subjects receiving a high protein diet as determined by both methods. CONCLUSIONS High protein diet is accompanied by increased stimulation of glucagon and insulin within the endocrine pancreas, high glycogen turnover and stimulation of gluconeogenesis.