Background: Level of Global DNA methylation is associated with many diseases and the influence of physical activity is being investigated by several research groups. The aim of our study was to assess the effect of lifetime endurance physical activity on global DNA methylation, physical fitness and body composition. Methods: A total of 31 elderly males were involved in the study, divided into two groups based on differences in physical activity. The first group consisted of 18 volunteers with lifetime endurance activity (mean age: 65.1 ± 3.3 yr.; height: 174.8 ± 4.9; weight: 81.5 ± 6,1 kg; BMI: 24.2 ± 1.1). The control group consisted of thirteen elderly individuals with a sedentary lifestyle (mean age: 64.8 ± 3.1 yr.; height: 176.5 ± 5.5; weight: 87.9 ± 10.1 kg; BMI: 27.8 ± 2.9). Quantification of global DNA methylation was performed in DNA isolated from peripheral blood mononucleated cells by LINE-1 pyrosequencing. Results: Elderly individuals with lifetime endurance activity had a better level of physical fitness VO2max on average 30 % (35.7 ± 10.6 vs. 31.9 ± 7,7 ml.kg-1.min-1, p <.01) and lower mean body fat content (17.46 ± 2.52 vs. 27.8 ± 2.9 %, p < .01). Global DNA methylation did not differ between studied groups (81.1 ± 2.1 vs. 80.5 ± 1.6 %). Conclusion: Better level of physical fitness does not influence the level of the global DNA methylation in peripheral blood mononuclear cells significantly. For future research we recommended to observe DNA methylation changes in specific tissues (e.g. skeletal muscle fibres).