Aims. Plasma levels of some biomarkers and markers of collagen turnover may reflect myocardial structural abnormalities associated with diastolic dysfunction. The aim of this study was to determine whether these markers could contribute to the diagnostics of heart failure with normal ejection fraction (HFNEF). Methods and Results. 91 patients with exertional dyspnea and normal left ventricular ejection fraction and 20 healthy controls underwent plasma analysis of markers of collagen turnover and other biomarkers, spirometry, and resting and exercise echocardiography. 38 patients with dyspnea had evidence of HFNEF, diagnosed at the early stage. Compared to the remaining patients, those with HFNEF had a significantly higher plasma levels of carboxy-terminal telopeptide of collagen type I (median 4.5 µg/L vs. 3.5 µg/L, P<0.05) and big endothelin (median 1.1 pmol/L vs 0.9 pmol/L, P<0.05). Univariate logistic regression analysis revealed a significant association between HFNEF and the following biomarkers: big endothelin, amino-terminal propeptide of type III procollagen (PIIINP), and matrix metalloproteinase-2 (MMP-2). However, none of these biomarkers independently contributed to the HFNEF diagnostics in a multivariate logistic regression analysis. Conclusion Plasma levels of big endothelin, PIIINP, and MMP-2 were found to be associated with the presence of early diagnosed HFNEF. However, none of these biomarkers contributed independently to current noninvasive HFNEF diagnostics recommended by the European Society of Cardiology guidelines.