Background Both aerobic exercise and whey protein can improve glucose regulation. The purpose of this study was to investigate how a single bout of vigorous-intensity aerobic exercise and whey protein, independently, as well as when combined, influence glycemia during an oral glucose tolerance test in sedentary, young men. Methods Healthy males ( n  = 11) completed four randomized trials: no exercise/no whey protein (R); exercise (EX; walking at 70% VO 2max for 60 min); 50 g of whey protein (W); and exercise combined with 50 g of whey protein (EXW). Each trial included a 75 g oral glucose tolerance test (OGTT) that was completed after an overnight fast. Blood samples were collected over a two-hour period during the OGTT. For EX and EXW, the exercise was performed the evening before the OGTT and the 50 g of whey protein was dissolved in 250 mL of water and was consumed as a preload 30 min prior to the OGTT. For R and EX, participants consumed 250 mL of water prior to the OGTT. Plasma samples were analyzed for glucose, insulin, C-peptide, glucagon, gastric inhibitory peptide (GIP) and glucagon like peptide 1 (GLP-1), and postprandial incremental area under the curve (iAUC) was calculated for each. Results Glucose iAUC was reduced during W (− 32.9 ± 22.3 mmol/L) compared to R (122.7 ± 29.8 mmol/L; p  < 0.01) and EX (154.3 ± 29.2 mmol/L; p  < 0.01). Similarly, glucose iAUC was reduced for EXW (17.4 ± 28.9 mmol/L) compared to R and EX ( p  < 0.01 for both). There were no differences in iAUC for insulin, C-peptide, GIP, GLP-1, and glucagon between the four trials. Insulin, C-peptide, glucagon, GIP, and GLP-1 were elevated during the whey protein preload period for W and EXW compared to EX and R ( p  < 0.01). There were no differences for insulin, C-peptide, glucagon, GIP, or GLP-1 between trials for the remaining duration of the OGTT. Conclusions Glucose responses during an oral glucose tolerance test were improved for W compared to EX. There were no additional improvemen The purpose of this study was to investigate how a single bout of vigorous-intensity aerobic exercise and whey protein, independently, as well as when combined, influence glycemia during an oral glucose tolerance test in sedentary, young men. Methods Healthy males ( n = 11) completed four randomized trials: no exercise/no whey protein (R); exercise (EX; walking at 70% VO 2max for 60 min); 50 g of whey protein (W); and exercise combined with 50 g of whey protein (EXW). Each trial included a 75 g oral glucose tolerance test (OGTT) that was completed after an overnight fast. Blood samples were collected over a two-hour period during the OGTT. Similarly, glucose iAUC was reduced for EXW (17.4 ± 28.9 mmol/L) compared to R and EX ( p < 0.01 for both). There were no differences in iAUC for insulin, C-peptide, GIP, GLP-1, and glucagon between the four trials. Insulin, C-peptide, glucagon, GIP, and GLP-1 were elevated during the whey protein preload period for W and EXW compared to EX and R ( p < 0.01). There were no differences for insulin, C-peptide, glucagon, GIP, or GLP-1 between trials for the remaining duration of the OGTT. Conclusions Glucose responses during an oral glucose tolerance test were improved for W compared to EX. In addition to timing, the dose or amount of whey protein consumed appears to influence the magnitude of its effects on blood glucose [ 21 ]. Several studies have demonstrated positive effects on insulin and glucose responses with higher doses (20 – 55 g) of whey protein [ 19 , 23 , 27 , 28 ]. Our lab has previously examined how differences in the dose of whey protein can influence glycemia. We found greater improvements in glycemic responses during a 75 g oral glucose tolerance test (OGTT) when 30 g of whey protein, compared to 20 g, was consumed 30 min prior to the OGTT [ 29 ]. When considering whey protein’s ability to increase insulin secretion and exercise’s effects on insulin sensitivity, we hypothesized that acute vigorous-intensity aerobic exercise (70% of VO 2max ; performed the previous day, 12 to 14 h before the whey protein consumption) in combination with a 50 g preload of whey protein prior to an oral glucose tolerance test would result in greater improvements in postprandial blood glucose responses when compared to whey protein or acute aerobic exercise alone. Methods Study population Twelve apparently healthy, sedentary males aged 18 to 44 years were recruited for this study. a Represents a significant difference compared to R . b Represents a significant difference compared to EX p < 0.01; Fig. 3 B). Additionally, glucose iAUC was lower for EXW (17.4 ± 28.9 mmol/L) compared to R ( p < 0.01) and EX ( p < 0.01). There were no differences between W and EXW. Insulin and C-peptide As shown in Fig. 4 A, insulin was elevated during the 30-min whey 2 ), there were no differences in insulin iAUC between the four trials. C-peptide response was similar to insulin, with a significant increase between timepoints -30 to 0 for W (1979.0 ± 260.1 pg/mL; p = 0.02) and EXW (1984.4 ± 273.6 pg/mL; p < 0.01) compared to EX (982.8 ± 160.6 pg/mL) and R (1183.7 ± 215.6 pg/mL; p < 0.01; Fig. 5 A). No differences were observed for C-peptide iAUC between the four trials (Fig. 5 B). Fig. 4 A Insulin response (pg/mL) prior to and during a two-hour oral glucose tolerance test between each trial. B Insulin iAUC (pg/mL) during the two-hour oral glucose tolerance test between each trial. There were no significant differences in iAUC between the four trials for glucagon (Fig. 7 B). Fig. 7 A Glucagon response (pg/mL) prior to and during a two-hour oral glucose tolerance test between each trial. B Glucagon iAUC (pg/mL) during the two-hour oral glucose tolerance test between each trial. R ; no exercise, no whey protein, EX; exercise, no whey protein, W ; no exercise, whey protein, EXW; exercise, whey protein. a and b represent significant differences for W compared to R and EX between timepoints -30 to 0, respectively.