Exercise during pregnancy or lactation may create a competition for glucose between the exercising muscle and either the developing fetus or the lactating mammary gland. To test these two hypotheses, pregnant rats were randomly assigned to isoenergetic diets with varying levels of glucose (20, 40 or 60% by weight) and fat (30, 22 or 14%, respectively, by weight) and were rested (R) or exercised (E) on a motorized treadmill at 20 m/min, 60 min/d (low intensity), 7 d/wk throughout pregnancy and lactation. Main effects and selected interactions of diet and exercise during pregnancy and diet, exercise and litter size during lactation were tested using 3 x 2 and 3 x 2 x 2 factorial designs, respectively. Neither diet nor exercise affected pregnancy outcomes. In contrast, during lactation, milk and mammary gland compositions and pup growth were altered. Exercise produced higher milk protein concentrations (40% glucose diet) and lower milk lactose concentrations (20% glucose diet). Exercise also lowered mammary gland fat content and produced higher milk fat concentrations. The 60% glucose diet resulted in the highest milk fat concentrations, but pups of dams fed the 40% diet were heavier on lactation d 15 than pups of dams fed the 60% diet. Taken together, these results support the claim of decreased availability of glucose to the mammary gland for lactose synthesis during chronic low intensity exercise. Additionally, the best lactation performance was not supported by a high carbohydrate (60% glucose), lower fat (14%) intake. A more moderate carbohydrate (40% glucose), higher fat (22%) intake promoted greater pup weights at weaning, suggesting an overlooked role for macronutrient composition in optimizing lactation performance.