Sterilization of rodent feed by steam autoclaving is a common practice in many research institutions. Often we only consider the beneficial effects of this process-the reduction of microbial contamination-and forget that the high temperatures and pressures can have negative effects on diet quality. The purpose of our study was to assess both the physical and chemical changes to a standard rodent feed autoclaved at multiple sterilization temperatures and the effects of the treated diets on mice. Pelleted NIH31 rodent feed was autoclaved at 4 sterilization temperatures (230, 250, 260, and 270 °F). Feed pellet hardness and the acrylamide concentrations of the diets were tested and compared with irradiated NIH31 feed. Study diets were fed to mice for 28 d, after which tissue samples were collected for analysis of acrylamide, glycidamide (the active metabolite of acrylamide), and genotoxicity. Both feed pellet hardness and acrylamide concentration increased with increasing sterilization temperatures; however, neither affected feed intake or body weight gain. Plasma acrylamide and glycidamide were significantly elevated only in mice fed NIH31 diet autoclaved at 270 °F compared with the irradiated feed, whereas urine acrylamide and glycidamide metabolites were significantly elevated in most autoclaved diets. Liver DNA adducts, which correlate with genotoxicity, were significantly elevated in all autoclaved diets compared with the irradiated diet. Institutions that autoclave their animal diets should carefully consider the temperatures necessary to achieve feed sterilization and the type of studies in which these autoclaved diets are used.