INTRODUCTION: The daily number of meals has an effect on postprandial glucose and insulin responses, which may affect substrate partitioning and thus weight control. This study investigated the effects of meal frequency on 24 h profiles of metabolic markers and substrate partitioning. METHODS: Twelve (BMI:21.6+/-0.6 kg/m(2)) healthy male subjects stayed after 3 days of food intake and physical activity standardization 2x36 hours in a respiration chamber to measure substrate partitioning. All subjects randomly received two isoenergetic diets with a Low meal Frequency (3x; LFr) or a High meal Frequency (14x; HFr) consisting of 15 En% protein, 30 En% fat, and 55 En% carbohydrates. Blood was sampled at fixed time points during the day to measure metabolic markers and satiety hormones. RESULTS: Glucose and insulin profiles showed greater fluctuations, but a lower AUC of glucose in the LFr diet compared with the HFr diet. No differences between the frequency diets were observed on fat and carbohydrate oxidation. Though, protein oxidation and RMR (in this case SMR + DIT) were significantly increased in the LFr diet compared with the HFr diet. The LFr diet increased satiety and reduced hunger ratings compared with the HFr diet during the day. CONCLUSION: The higher rise and subsequently fall of insulin in the LFr diet did not lead to a higher fat oxidation as hypothesized. The LFr diet decreased glucose levels throughout the day (AUC) indicating glycemic improvements. RMR and appetite control increased in the LFr diet, which can be relevant for body weight control on the long term. TRIAL REGISTRATION: ClinicalTrails.gov NCT01034293.