We have demonstrated that postmenopausal women with endothelial nitric oxide synthase (eNOS) gene polymorphism at position -786T>C are less responsive in lowering blood pressure after exercise training than those who are not polymorphic (TT genotype),[1]. This response was accompanied by lower nitric oxide (NO) production, measured by plasma nitrite/nitrate. Interestingly, variable number of tandem repeat Intron 4b/a of the eNOS gene has also been associated with reduction in plasma NO levels, decreased eNOS expression or reduced enzyme activity[2]. However, no studies exist evaluating the influence of Intron 4b/a of the eNOS gene in the cardiovascular responses and oxidative stress biomarkers in response to aerobic exercise training (ET) in adults. Therefore, we investigated the relationship between eNOS polymorphism at position Intron 4 a/b and the effect of ET on the blood pressure (BP), nitrate/nitrite levels (NOx-) and oxidative stress biomarkers (superoxide dismutase – SOD, catalase – CAT and malondialdehyde – MDA) in middle-aged adults. Eighty-one subjects (51.1±0.6 yrs) were trained in treadmill, 3 days/week, for 30-40 min/session at moderate intensity, during 24 sessions. A comparison between sphygmomanometer-based and ambulatory BP monitoring (ABPM) was carried out before and after ET. Genotype analysis was performed using sequencing method (ABI3500-Applied Biosystems). All the data acquisition and analysis were performed in a double-blind manner. The volunteers were divided into non-polymorphic (Intron 4bb, n=55) and polymorphic (Intron 4ba+aa, n=26). ET were effective in lowering BP measured by sphygmomanometer-based for both systolic (-4.6mmHg) and diastolic BP (-2.6 mmHg) whereas in ABPM measurements only systolic BP values were significantly reduced (-2.4mmHg) in non-polymorphic group (genotype bb). These beneficial effects were accompanied by increased SOD (+11.7 U/ml) and CAT (+7.9 nmol/ml/min) enzymes activity. In contrast, adults who carry eNOS gene polymorphism for intron 4 a/b (genotype 4ba+aa) showed no changes in BP for both methods, acute and measurement during 24 hr. Interestingly, an increase in SOD (+12.1 U/ml) and CAT (+10.2 nmol/ml/min) enzymes activity were found in polymorphic group. No changes were observed in plasma NOx- and MDA concentrations in response of ET for both genotypes (intron 4bb and 4ba+aa). Our findings show that the polymorphism of eNOS gene at intron 4b/a position attenuated the benefits of the exercise training in lowering blood pressure, but did not affect the antioxidant enzymes activity.