The purpose of this research was to assess the amount of isoprostane 8-iPF2α-III, a marker of oxidation stress, in excreted urine. The study was conducted on 40 male rats aged 5 months. Group 1 received the basic diet, groups 2-4, the modified diet in which whole cereal grains were partly substituted with wheat flour and sucrose. Groups 1-2 received water to drink, groups 3 and 4 were given aqueous solutions of B group vitamins at different doses. On the fifth week of the experiment, a 24-h urine collection was conducted. The concentrations of creatinine and 8-iPGF2a-III were determined in urine samples. Modification of diet composition caused increased excretion of isoprostane, which may indicate enhancement of free-radical reactions in the examined animal. The provided supplementation decreased the value of this parameter expressed as a creatinine ratio, but not, however, to the values observed in the animals fed the basic diet. A stronger effect was observed with excess supplementation.