Fresh oil was heated for 20 h at 180°C with amino acids or gluten. A powdered diet (AIN93G; no fat) was mixed with 7 wt% of fresh oil (control), or supernatants of the heated oils described above, and fed to male Wistar rats for 12 weeks. No gross symptoms attributable to the heated oils were observed, but the gluten group showed a slow body weight increase; a significant difference was found in the weight after age 21 weeks in spite of diet consumption comparable to that of the control group. The serum of the heated oil groups showed a tendency toward lower values on various hematological measures, especially triacylglycerol and free fatty acid and toward higher values on aspartate aminotransferase (AST) and alanine aminotransferese (ALT), than those of the control group. All the rats except one in the gluten group had the same level of AST as those of the control rats, while the amino acid group included four rats with AST over 100 IU/L, the highest value in the control group. The number of dark red patches found on the surface of the liver and histological evaluation also showed frequent damage to the livers of the amino acid group. The difference in toxicity between the two heated oils seems to be related to the molecular sizes of amino acids and gluten. Gluten or melanoidin produced during heating probably decreased or counteracted the cytotoxicity of thermally oxidized oil. It is expected that oil heated with gluten can be used as a safe and effective oil for humans on weight-reduction diets.