Autoclaving is the current method of choice for sterilization of diets for specified-pathogen-free (SPF) laboratory animals, but this process may negatively affect the nutrient content (e.g., protein, vitamin) of the diets. Two natural-ingredient diets supplemented with either soyabean meal containing phytoestrogens (S) or with casein (C), and a commercial autoclavable (SN) diet, were autoclaved at 121°C for 20 min (T1) or at 134°C for 10 min (T2). Chemical composition, selected water- and fat-soluble vitamins, and acrylamide were analysed in all non-autoclaved (T0) and autoclaved diets, whereas in S diets, oestrogenic isoflavones were also determined. Autoclaving affected most protein bound to NDF macronutrient contents only to a small degree, except those of NDF and N-NDF. The contents of NDF and N-NDF were increased by T2 and T1; the change was the largest in the SN and the smallest in the C diet. Losses of particular vitamins differed among the diets and treatments. Among the water-soluble vitamins, thiamine, riboflavin and pyridoxine were the least affected, whereas calcium pantothenate was lowered by T1 and T2. Among fat-soluble vitamins, vitamin E was the most stable, while vitamins A and D, the least stable, but even the highest vitamin losses did not exceed 50%. The acrylamide concentration increased more in diets autoclaved at T2 than at T1 and in the S and C diets than in SN. Autoclaving the S diet increased the daidzin and genistin contents and slightly reduced the total isoflavone content. It is concluded that the effects of autoclaving different diets are not uniform, but longer autoclaving at a lower temperature (T1) is less detrimental than shorter treatment at a higher temperature (T2), mainly because of the smaller increase in the NDF and N-NDF contents and acrylamide concentration.