This research aimed to study the long-term effects of soccer training on platelet membrane fatty acid levels and antioxidant vitamins. Forty-four subjects divided into soccer players (SP; n = 22; 20.86 ± 0.36 years) and a control group (CG; n = 22; 21.23 ± 0.49 years) participated in the study. The fatty acids of the platelet membrane, the rates of desaturation, lipid peroxidation indexes and intra-platelet levels of vitamins C and E were assessed. SP obtained lower values in polyunsaturated fatty acids 18:3:3 (alpha-linolenic acid), 20:5:3 (eicosapentaenoic acid) and 22:6:3 (docosahexaenoic acid) (p < 0.05). The desaturation index ∆5 was higher in SP (p < 0.05), and they had a higher lipid peroxidation index 20:4:6 (arachidonic acid)/16:0 (palmitic acid) (p < 0.05). Vitamin E and C platelet values were also higher in SP (p < 0.01). There were positive correlations in the ω6/ω3 index (p < 0.05), desaturation index ∆5 (p < 0.05), lipid peroxidation index 20:4:6/16:0 and intra-platelet vitamins E and C (p < 0.01) with the level of physical activity. In addition, there were inverse correlations in fatty acids 24:0 (lignoceric acid), 16:1 (palmitoleic acid), 20:3:6 (eicosadienoic acid) and 18:3:3 (alpha-linolenic acid) (p < 0.05) depending on the degree of physical activity. Regular long-term soccer training could modify the concentration of fatty acids such as 24:0, 16:1, 18:6, 20:3:6, 18:3:3:3, 20:5:3, 26:6:3 and ω3 PUFAs in the platelet membrane.