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/m2) healthy male subjects stayed after 3 days of food intake and physical activity standardization 2×36 hours in a respiration chamber to measure substrate partitioning. All subjects randomly received two isoenergetic diets with a Low meal Frequency (3×; LFr) or a High meal Freque PLoS One PLoS ONE 440 plosone plos PLoS ONE 1932-6203 PLOS PMC3374835 PMC3374835.1 3374835 3374835 22719910 10.1371/journal.pone.0038632 PONE-D-12-01147 1 Research Article Biology Anatomy and Physiology Digestive System Digestive Physiology Digestive Regulation Endocrine System Endocrine Physiology Hormones Insulin Physiological Processes Energy Metabolism Homeostasis Social and Behavioral Sciences Psychology Sensory Perception Effects of Meal Frequency on Metabolic Profiles and Substrate Partitioning in Lean Healthy Males Effects of Meal Frequency Munsters Marjet J. M. * Saris Wim H. M. 2012 https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. 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 2×36 hours in a respiration chamber to measure substrate partitioning. All subjects randomly received two isoenergetic diets with a Low meal Frequency (3×; LFr) or a High meal Frequency (14×; HFr) consisting of 15 En% protein, 30 Energy intake is influenced by the effect of food’s energy density, total energy content and meal frequency and the extent to which these alter satiety. Of these factors, meal frequency has received least attention [1] . Epidemiological evidence indicates increasing trends in recent years of dietary snacking and increased meal frequency [2] , [3] . The current literature is mixed with regard to the efficacy of increased meal frequency (or snacking) regimens in causing metabolic alterations, particularly in relation to weight management [1] . Given the inconclusive evidence in the literature regarding meal frequency and its metabolic implications, 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. Materials and Methods The protocol for this trial is available as supporting information; see Protocol S1 . 10.1371/journal.pone.0038632.t001 Table 1 Subject characteristics at baseline. Mean, maximum, minimum glucose concentration, and the (net i)AUCs were calculated per diet per 24 hour from the CGMS data, during the day and night and showed no significant differences between the two interventions ( table 3 ). Nevertheless, the CGMS data clearly showed the different glycemic patterns of the two meal frequency diets ( figure 3 ). Glycemic variability (conga 1,2,4 and CV) did not change between both intervention diets ( table 3 ). The correlation between the CGMS data and glucose data was significant in the LFr diet (R 2 = 0.333; p = 0.05), and not in the HFr diet. greater peaks and lower troughs) 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. On the contrary, studies examining nibbling (small, frequent meals) compared to gorging (large, few meals) under isoenergetic conditions over a range of meal frequencies from 2 to 12 meals/d provided conflicting evidence, but over a narrower range suggest there may be some tendency for a 6-meals/d pattern to improve appetite control relative to a 3-meals/d pattern [34] . A point to consider when interpreting the study findings includes the energy level of the study diets (varied from energy restriction to isoenergetic) and resulting meal portions. In conclusion, glucose and insulin profiles showed greater fluctuations, but a lower AUC of glucose in the LFr diet compared with the HFr diet. The higher peaks and subsequently lower troughs of insulin in the LFr diet did not lead to a higher fat oxidation as hypothesized. RMR and appetite control increased in the LFr diet, which can be relevant for body weight control on the long term. However, this was studied for one day in young healthy males, which are very metabolic flexible.