N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) level at rest is related to left ventricular (LV) function and cardiovascular mortality in patients with heart failure (HF). There are limited and controversial data regarding changes in NT-pro-BNP level during exercise in patients with HF. The aim of this study was to investigate the effects of exercise on NT-pro-BNP levels and the relation between increases in NT-pro-BNP and the LV ejection fraction and cardiovascular mortality in patients with HF. Seventy-five patients with HF (New York Heart Association classes I to III) and 20 healthy subjects were enrolled in the study. Echocardiographic examination was performed. The modified Bruce protocol was used for symptom-limited exercise testing. Levels of NT-pro-BNP were measured at rest and after peak exercise. The patients were followed up for 690 to 840 days for cardiovascular mortality. Exercise induced significant increases in NT-pro-BNP in patients and controls. Except for a relative increase in NT-pro-BNP during exercise (relative DeltaNT-pro-BNP), NT-pro-BNP concentrations at rest and during peak exercise and absolute increases in NT-pro-BNP during exercise (absolute DeltaNT-pro-BNP) were significantly higher in patients with HF (p <0.001). Absolute DeltaNT-pro-BNP was positively correlated with NT-pro-BNP at rest (p <0.001). The level of absolute DeltaNT-pro-BNP was the most important parameter in predicting a LV ejection fraction <30% (p <0.001). Absolute DeltaNT-pro-BNP and LV end-systolic volume were found to be independent predictors of mortality (p = 0.012 and p = 0.015, respectively). In conclusion, exercise induced increases in NT-pro-BNP in patients and healthy subjects. Absolute increase in NT-pro-BNP is a reliable parameter in predicting a low LV ejection fraction and may help in the identification of patients at high risk for mortality.