Cardiac disease in diabetes presents as impaired left ventricular contraction and relaxation; however, the mechanisms underlying contractile protein dysfunction during the progression of disease are unknown. Accordingly, we assessed Ca²⁺-dependent tension development and tension-dependent ATP consumption (tension cost) in a rat model early (6 wk) and late (12 wk) after the onset of diabetes (50 mg/kg iv streptozotocin) using mechanical force- and enzyme-coupled UV absorbance measurements. Myofilament Ca²⁺ sensitivity and maximal tension were unchanged between groups at either time point. Cross-bridge cycling rate was significantly decreased in diabetes, as indexed by tension cost (early control 5.4 ± 0.4 and early diabetes 4.2 ± 0.3; and late control 6.0 ± 0.2 and late diabetes 4.2 ± 0.2; P < 0.05). Because rodent models of cardiac disease are confounded by altered myosin isoform distribution, myosin content was determined by SDS-PAGE and densitometry. The cardiac content of α-myosin in diabetes was decreased to 41% ± 4.1 at 6 wk and 32.5% ± 2.9 at 12 wk of diabetes (early control 77.8% ± 3.3 and late control 73.6% ± 2.5). Separate control experiments demonstrated a linear decrease in tension cost with decreased α-myosin content. Given this, the depression of tension cost in this rodent model of diabetes could be fully explained by the altered myosin isoform distribution.