The potential confounding effect of different amounts and proportions of macronutrients across eating patterns on meal or dietary glycemic index (GI) and glycemic load (GL) value determinations has remained partially unaddressed. The study aimed to determine the effects of different amounts of macronutrients and fiber on measured meal GI and GL values. Four studies were conducted during which participants [ = 20-22; women: 50%; age: 50-80 y; body mass index (in kg/m): 25-30)] received food challenges containing different amounts of the variable nutrient in a random order. Added to the standard 50 g available carbohydrate from white bread was 12.5, 25, or 50 g carbohydrate; 12.5, 25, or 50 g protein; and 5.6, 11.1, or 22.2 g fat from rice cereal, tuna, and unsalted butter, respectively, and 4.8 or 9.6 g fiber from oat cereal. Arterialized venous blood was sampled for 2 h, and measured meal GI and GL and insulin index (II) values were calculated by using the incremental area under the curve (AUC) method. Adding carbohydrate to the standard white-bread challenge increased glucose AUC ( < 0.0001), measured meal GI ( = 0.0066), and mean GL ( < 0.0001). Adding protein (50 g only) decreased glucose AUC ( = 0.0026), measured meal GI ( = 0.0139), and meal GL ( = 0.0140). Adding fat or fiber had no significant effect on these variables. Adding carbohydrate (50 g), protein (50 g), and fat (11.1 g) increased the insulin AUC or II; fiber had no effect. These data indicate that uncertainty in the determination of meal GI and GL values is introduced when carbohydrate-containing foods are consumed concurrently with protein (equal amount of carbohydrate challenge) but not with carbohydrate-, fat-, or fiber-containing foods. Future studies are needed to evaluate whether this uncertainty also influences the prediction of average dietary GI and GL values for eating patterns. This trial was registered at clinicaltrials.gov as NCT01023646.