The potential impact of prior meal composition on the postprandial glycemic response and glycemic index (GI) and glycemic load (GL) value determinations remains unclear. We determined the effect of meals that varied in macronutrient composition on the glycemic response and determination of GI and GL values of a subsequent standard test food. Twenty healthy participants underwent 6 test sessions within 12 wk. The subjects received each of 3 isocaloric breakfast meals (i.e., high carbohydrate, high fat, or high protein) on separate days in a random order, which was followed by a standard set of challenges (i.e., white bread and a glucose drink) that were tested on separate days in a random order 4 h thereafter. Each challenge provided 50 g available carbohydrate. Arterialized venous blood was sampled throughout the 2-h postchallenge period. GI, GL, and insulin index (II) values were calculated with the use of the incremental area under the curve (AUC) method, and serum lipids were determined with the use of standard assays. The consumption of the high-protein breakfast before the white-bread challenge attenuated the rise in the postprandial serum glucose response ( < 0.0001) and resulted in lower glucose AUC ( < 0.0001), GI ( = 0.0096), and GL ( = 0.0101) values than did the high-carbohydrate and high-fat breakfasts. The high-protein breakfast resulted in a lower insulin AUC ( = 0.0146) for white bread than did the high-fat breakfast and a lower II value ( = 0.0285) than did the high-carbohydrate breakfast. The 3 breakfasts resulted in similar serum lipid responses to the white-bread challenge. These data indicate that the macronutrient composition of the prior meal influences the glycemic response and the determination of GI and GL values for white bread. Future studies are needed to determine whether the background food macronutrient composition influences mean dietary GI and GL values that are calculated for eating patterns, which may alter the interpretation of the associations between these values and chronic disease risk. This trial was registered at clinicaltrials.gov as NCT01023646.