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Fructose, which is a sweetener with a low glycemic index, has been shown to elevate postprandial triglyceride compared with glucose. There are limited data on the effect of fructose in a solid mixed meal containing starch and protein.We determined the effects of sucrose, fructose, and sucralose on triglyceride, glucose, and insulin in an acute study in healthy, overweight, and obese individuals.The study had a randomized crossover design. Twenty-seven participants with a mean age of 44 y and a mean body mass index (in kg/m(2)) of 26 completed the study. Fructose (52 g), sucrose (65 g), and sucralose (0.1 g) were delivered as sweet-taste-balanced muffins with a total fat load (66 g). Blood samples were taken at baseline and every 30 min for 4-h glucose, triglyceride, and insulin concentrations, and the area under the curve (AUC) and the incremental area under the curve (iAUC) were analyzed.No significant difference was shown between the 3 sweeteners for triglyceride and glucose concentrations and the AUC. The glucose iAUC was lower for fructose than for sucrose and sucralose (P < 0.05). Insulin concentrations differed significantly by the type of muffin (P = 0.001), the interaction of time by type of muffin (P = 0.035), the AUC (P < 0.001), and the iAUC (P < 0.001). Fructose had a significantly lower insulin response than that of either sucrose (P-treatment = 0.006) or sucralose (P-treatment = 0.041).Fructose, at a moderate dose, did not significantly elevate triglyceride compared with sucrose or sucralose and lowered the glucose iAUC. These results indicate that these sweeteners, at an equivalent sweetness, can be used in normal solid meals. Fructose showed a lower insulin response, which may be beneficial in the long term in individuals at risk of type 2 diabetes. This trial was registered at the Australian New Zealand Clinical Trials Registry as ACTRN12615000279527.