The current literature is mixed with regard to the efficacy of increased meal frequency regimens in causing metabolic alterations, particularly in relation to weight management. Increasing eating frequency has been postulated to increase metabolism, reduce hunger and food cravings, improve glucose and insulin control, and reduce body weight and body fat storage. However, there are suggestions from experimental studies to date as well as from cross-sectional epidemiological studies, in which energy intake underreporting is taken into account, that greater eating frequency may promote positive energy balance in free-living adults. On the other hand, well-controlled intervention studies do not support an association between eating frequency and body weight. Eating three meals a day is suggested to result in a higher postprandial insulin peak due to the higher carbohydrate intake and thereby increasing cellular glucose uptake and oxidation. As a consequence, dietary fat is primarily stored in the adipose LY294002 PI3K inhibitor tissue during the postprandial phase. In between meals, the fasting state, when insulin levels are decreased and lipolysis is activated this substrate flux is reversed. Very well-controlled trials are necessary to resolve speculation that the current increase in snacking habits contribute by its metabolic changes during the day to the escalating obesity epidemic. For that reason, the aim of the present study was to investigate the mechanistic effects of meal frequency on 24 hr insulin, glucose profiles, appetite profiles and substrate partitioning under well-controlled energy balance conditions. We hypothesized that in an energy balanced situation eating 3 meals a day gives better opportunities to turn the metabolic flux into a prolonged fasting state with a higher fat oxidation compared to eating 14 meals a day where subjects remain in a continuous postprandial status. Increasing meal frequency resulted in significantly lower peaks, higher troughs and constant glucose and insulin values compared with the LFr diet under isoenergetic wellcontrolled conditions in lean healthy males. Nevertheless, no effect of meal frequency was observed on substrate partitioning of CHO and fat. Protein oxidation, RMR and appetite control increased significantly in the LFr diet compared with the HFr diet. Our results are in accordance with findings from Solomon et al., who found that 2 meals per day led to greater fluctuations in glucose, insulin, and ghrelin responses compared with the 12 meals per day assessed throughout an 8-h period. Nevertheless, the lower AUC of glucose in the LFr indicates glycemic improvements, we suggest that this can lead to a better body weight control on the long term. The CGMS data showed the glycemic excursions and clearly indicated the differences between the two diets during the day. However, baseline values are somewhat lower than the glucose levels measured at the fixed time points. The accuracy of the sensor has been discussed and discrepancies occasionally were seen between interstitial tissue and blood glucose levels in detecting low glucose values.