The clinical use of doxorubicin, an effective chemotherapeutic is hampered by the development of irreversible cardiotoxicity. Here we test time-frequency analysis of heart rate (HR) variability (HRV) for early detection of doxorubicin-induced cardiotoxicity. Experiments were conducted in adult male Wistar rats treated for 15 days with doxorubicin (DOXO, total dose 15 mg kg(-1), i.p.) or saline (CONT). DOXO rats exhibited cardiotoxicity confirmed by histological examination without developing heart failure as estimated by echocardiography. However, HR variability increase reflected subtle microscopic changes of cardiac toxicity in DOXO rats. The results recommend time-frequency analysis of HRV for early detection of doxorubicin-induced cardiomyopathy.