The aims of this study were to investigate the effects of repeated and multiple-dose pimobendan on cardiac systolic function and the correlations between changes in cardiac systolic function and plasma concentrations of pimobendan and O-desmethylpimobendan (ODMP). Five clinically healthy cats were subjected to four different medication protocols for 14 days, with a washout period of at least 1 month between each protocol. The protocols were pimobendan 0.5 mg/kg q12h (high dosage [HD] group); pimobendan 0.25 mg/kg q12h (standard dosage [SD] group); pimobendan 0.125 mg/kg q12h (low dosage group); and Biofermin R, one tablet q12h (placebo group). Echocardiography and measurement of plasma concentrations of pimobendan and ODMP were performed prior to medication administration (baseline) and 20, 40, 60, 120, 240, 360, and 480 min after administration, and the correlation between the changes in cardiac systolic function and plasma concentration of pimobendan, ODMP, or the sum of both were examined. The cardiac systolic function increased in the HD and SD groups, and there were significant correlations between the lateral peak systolic myocardial velocity (S′) changes and plasma pimobendan, plasma ODMP, and the sum of both. Repeated doses of pimobendan in healthy cats increased cardiac systolic function, and there were significant correlations between cardiac function and plasma concentrations of pimobendan and ODMP. The results of this study highlight the effectiveness of a higher dose of pimobendan.