The incidence of type 2 diabetes worldwide has increased significantly over the past decades, which is associated with changing dietary habits and physical inactivity. According to the diet, so far there has been a great focus on the quality of carbohydrates and fat in relation to metabolic health, while the importance of protein has been neglected. The Danes' average protein intake is 1.5 g/kg/day, which is at the high end of the recommendations (0.8-1.5 g/kg/day) from the Nordic Nutrition Recommendations (NNR 2023). Recent studies in rodents have shown that protein restriction has positive effects on health, including improved glucose and insulin homeostasis and reduced fat mass, while a high intake of protein has a negative effect on insulin sensitivity. Previously the investigators have shown, in healthy young men, that consuming a diet low in protein (0.9 g/kg/day), compared to the participants usual diet (1.5 g/kg/day), over 7 days, resulted in an increased insulin sensitivity as well as a marked increase in the plasma fibroblast growth factor 21 (FGF21) concentration. The increased insulin sensitivity is thought to be mediated by the increase in plasma FGF21 concentration. However, the effect is not yet fully understood. It is also not clear whether the increase in plasma FGF21 concentration, as well as the mentioned metabolic effects on insulin and glucose homeostasis, will take place if the participants are kept weight stable on a eucaloric diet.

A three-arm protocol was performed. In protocol 1, participants ingested either a low-protein (LP) meal or a higher protein (HP) meal in a randomized order, separated by 72 h, followed by a 5-week low-protein, high-carbohydrate (LPHC) diet. In protocol 2 and 3, participants ingested either a LPHC diet or a low-protein, high-fat (LPHF) diet for 5 weeks followed by a higher protein diet (HPD) for another 5 weeks. All diets were eucaloric. Resting metabolic rate (RMR) was measured pre, during and post the protein-restricted interventions. A hyperinsulimic-euglycemic clamp was performed post the protein-restricted interventions and post the HPD interventions. A basal subcutaneous abdominal fat biopsy was obtained post the protein-restricted interventions and post the HPD interventions. In a run-in period over 14 days, the participant records the daily number of steps and physical activity, in order to determine their habitual activity level. During this period, a 4-day dietary record is performed, where all ingested food was weighed and recorded. During the experimental period the participant followed the supplied diet. All food consumed during the interventions was handed out to the subject at the institute. All food was packaged and weighed to 1 gram of accuracy. During the dietary intervention, a weekly test in the morning was performed, where the resting metabolic rate was measured and blood samples taken from the arm vein. At week 0, 5 and week 10 body composition was measured by dual energy x-ray absorptiometry scanning (DXA), and the fat biopsy was obtained . At all tests participants arrived fasting at the institute at 8:00 a.m. in the morning. The participant arrived by car or public transport.

Inclusion Criteria:

• BMI≤25
• Maximal oxygen uptake ≤42-51 ml/kg/min) • non-smoking

Exclusion Criteria:

• The use of medicine