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Background & aims: This study characterized the glucose and insulin responses to temporal alterations in meal frequency, and alterations in the macronutrient composition. Methods: Eight subjects underwent three separate 12-h meal tests: three high carbohydrate (3CHO) meals, 6 high carbohydrate meals (6CHO), 6 high-protein meals (6HP). Blood samples were taken at 15-min intervals. Integrated area under the curve (AUC) concentrations for glucose and plasma insulin were determined (total, 4-h, and 2-h blocks) for each meal condition. Results: Baseline glucose and insulin values were not different between study days. Peak glucose levels were highest on the 3CHO day; however the 12 h glucose AUC was higher during the 6CHO condition (p = 0.029) than 3CHO condition, with no difference in the insulin response. The 6HP condition resulted in a decreased glucose AUC (p = 0.004) and insulin AUC (p = 0.012) compared to 6CHO. Conclusions: In non-obese individuals, glucose levels remained elevated throughout the day with frequent CHO meals compared to 3CHO meals, without any differences in the insulin levels. Increasing the protein content of frequent meals attenuated both the glucose and insulin response. These findings of elevated glucose levels throughout the day warrant further research, particularly in overweight and obese individuals with and without type 2 diabetes