BACKGROUND: The relationships between diet, exercise, and prostate cancer (PCa) remain unclear. We have previously reported that a "Western" diet promotes PCa tumor growth in vivo. Presently, we report the effects of sustained aerobic exercise on PCa progression in animals fed a high-fat diet versus a standard diet. METHODS: Athymic mice (n = 43) were inoculated subcutaneously with human PCa (LNCaP) cells, fed ad libitum with either a high-fat or a standard diet, and randomized into forced exercising and non-exercising groups. Body weight, tumor volume, and food consumption were recorded tri-weekly. Terminal serum samples and tumor biopsies were obtained for analysis. RESULTS: Body weight differences were not observed between the groups over time. The high-fat diet with exercise (HF-Ex) group showed significantly increased tumor growth rate compared to all other groups (P < 0.0007). Tumor growth rate of the standard diet with exercise (Std-Ex) group was reduced significantly compared to the high-fat diet without exercise (HF-No Ex) group (P = 0.0008). Significant differences (P ≤ 0.012) were observed in energy consumption (kcal) between the groups over time. Exercising mice consumed significantly more kcal than non-exercising mice, and the HF-Ex group consumed significantly more than each of the other three groups (P < 0.0007). The expression levels of p27 and p21 were increased in exercising animals, while AR expression was elevated in the HF-Ex group versus the Std-Ex and HF-No Ex groups. CONCLUSIONS: Sustained aerobic exercise did not counteract the tumor-promotional effect of increased consumption of a high-fat diet, suggesting that diet is more influential in PCa progression than exercise. Combining exercise with a healthy diet reduced the rate of PCa progression in this model. This study may have implications for PCa risk reduction in humans. Prostate 9999:XX-XX. © 2013 Wiley Periodicals, Inc.