The effects of non-fried and fried virgin olive and sunflower oils on rat liver microsomal compositional features have been investigated. In addition, plasma antioxidants (α-tocopherol and ubiquinone 9) were investigated as well as the possible oxidative modifications suffered by virgin olive and sunflower oils during the frying process. The frying process decreased the content of α-tocopherol and phenolics in the oils and increased total polar materials. Sunflower oil was affected to a greater extent than olive oil. In rats, the intake of fried oil led to higher levels of lipid peroxidation and a lower concentration of plasma antioxidants. Microsomal fatty acid and antioxidant profiles were also altered. It seems that a strong relationship exists between the loss of antioxidants and the production of toxic compounds in the oils after frying and the extent of the peroxidative events in microsomes, which were also different depending on the fat source. The highly unsaturated sunflower oil was less resistant to the oxidative stress produced by frying and led to a higher degree of lipid peroxidation in liver microsomes in vivo than virgin olive oil.