As studying with population carrying no classical cardiovascular risk factors seems to be an advantage in isolating effects of regular exercise on endothelial functions, inflammatory and thrombotic activity; the present study was designed to evaluate the clear effects of long-term regular exercise in middle-aged, healthy men. A total of 32 regularly exercising (three times per week, 12.8 +/- 6.8 years) men (Group I, mean age = 53.2 +/- 6.1 yrs) and 32 sex- and age-matched sedentary subjects (Group II, mean age = 51.0 +/- 7.7 yrs) were involved in the study. All participants were non-smokers and with no history of hypertension and diabetes. During one day preceding tests, the subjects refrained from training and maintained their normal diet. In all subjects, body mass index (BMI), percentage of body fat (% BF) and maximal oxygen uptake (VO2max) were calculated. Serum uric acid, glucose, HbA1c, lipids, high-sensitive C-reactive protein (hs-CRP), fibrinogen levels, white blood cell (WBC) and platelet count were measured. Resting heart rates and blood pressures were recorded and standard exercise stress test was applied using the modified Bruce protocol. Flow-mediated and nitrate-induced dilatation (FMD and NID) of the brachial artery and carotid intima-media thickness (cIMT) were evaluated as markers of endothelial functions and early atherosclerosis. Mean BMI, % BF, systolic and diastolic blood pressures, WBC and platelet count, HbA1c, total and LDL cholesterol, hs-CRP and fibrinogen levels were similar between the groups. Group I had significantly lower serum glucose, uric acid and triglyceride (p < 0.05, p < 0.005 and p < 0.05, respectively) and higher HDL cholesterol levels (p < 0.0001) than in Group II. FMD values were significantly higher in Group I than in Group II (p < 0.005) while there were no significant differences in NID and cIMT measures between the groups. VO2max and cIMT showed a negative correlation in Group I (r = -0.463, p < 0.0001). Negative correlations also existed between VO2max and fibrinogen levels in both Group I and II (r = -0.355, p < 0.05 and r = -0.436, p < 0.05, respectively). These results are concordant with the concept of favorable effects of regular physical exercise on cardiovascular health based on enhancement of endothelial functions even in subjects who have low cardiovascular risk profile.