Osmotic fragility test has been used to study the influence of different factors on the osmotic properties of cell membranes. Considering the importance of polyunsaturated fatty acids (PUFA) in biological functions we aimed to investigate the effects of dietary PUFA supplementation on erythrocyte osmotic fragility (EOF), blood lactate (BL), hematocrit (Hct), red blood cell (RBC), hemoglobin (Hb), and mean cell volume (MCV) in 10 jumper horses. Two events occurred prior to start supplementing horse’s diet and two events occurred after 4 weeks PUFA supplementation. Five horses received the PUFA supple- mentation (Group A), and five served as controls (Group B). Blood samples were taken before and after each course. The statistical analysis revealed significant increase in BL, Hct, RBC and Hb following ex- ercise (Po0.0001). However, the interaction between exercise and PUFA supplementation (P1⁄40.0083) showed PUFA-supplemented horses having a smaller rise in BL levels (P1⁄40.0107) following exercise. Significant interactions between exercise and PUFA treatment were also found on EOF levels (Po0.05). The hemolysis curves showed PUFA-supplemented horses exhibiting a reduction in EOF compared to controls (Po0.05). Although hemolysis never occurred at 0.9% NaCl concentration, jumping exercise determined an increase in EOF (P1⁄40.0014) at 0.8% NaCl solution. A significant interaction between ex- ercise and PUFA treatment (P1⁄40.0022) was found showing PUFA-supplemented horses having lower EOF (P1⁄40.0015) following exercise. The assessment of EOF is a suitable indicator of athletic performance. The results showed that PUFA supplementation might exert beneficial effects on the horse body system by enhancing the performance in high-level show jumpers.