BACKGROUND: Currently, limited information is available regarding testosterone in women with heart failure with preserved ejection fraction (HFpEF). This cross-sectional study aimed to elucidate the clinical significance of serum total testosterone concentration (T-T) in Japanese elderly women with pre-HFpEF, a condition that develops before the onset of HF. METHODS: A total of 232 elderly women outpatients with pre-HFpEF (mean age ± standard deviation, 75 ± 7 years) were enrolled. Relationships between T-T and other clinical parameters were investigated. The definition of pre-HFpEF in this study was that patients with left ventricular ejection fraction of 50% or more and E/e' ratio as a marker of left ventricular function greater than 9 on echocardiography had no HF symptoms and no history of HF hospitalization. RESULTS: There was a significantly negative correlation between T-T and the E/e' ratio (r = -0.24, P < 0.001), and low T-T was significantly related to coronary artery disease. A significant correlation was observed between T-T and biomarkers such as blood brain natriuretic peptide concentration (r = -0.31, P < 0.001), serum high-sensitivity cardiac troponin T concentration (r = -0.33, P < 0.001), and the derivatives-reactive oxygen metabolites test, an oxidative stress marker (r = -0.46, P < 0.001). Furthermore, multiple regression analysis identified that the factors described above were selected as independent variables for T-T as a dependent variable. CONCLUSIONS: This cross-sectional study indicated that low T-T levels reflect unfavorable pathophysiological conditions in Japanese elderly women with pre-HFpEF. To clarify the relevance of T-T as a predictive indicator for the onset of cardiovascular diseases, including HF incidence in elderly women with pre-HFpEF, future prospective studies, including interventional treatments, should be conducted.