Chronic pressure-overload and diabetes mellitus are two frequent disorders affecting the heart. We aimed to characterize myocardial structural and functional changes induced by both conditions. Pressure-overload was established in Wistar-han male rats by supra-renal aortic banding. Six-weeks later, diabetes was induced by streptozotocin (65mg/kg,ip), resulting in four groups: SHAM, banding (BA), diabetic (DM) and diabetic-banding (DB). Six-weeks later, pressure-volume loops were obtained and left ventricular samples were collected to evaluate alterations in insulin signalling pathways, extracellular matrix as well as myofilament function and phosphorylation. Pressure-overload increased cardiomyocyte diameter (BA 22.0±0.4μm, SHAM 18.2±0.3μm) and myofilament maximal force (BA 25.7±3.6kN/m(2), SHAM 18.6±1.4kN/m(2)), Ca(2+) sensitivity (BA 5.56±0.02, SHAM 5.50±0.02) as well as MyBP-C, Akt and Erk phosphorylation, while decreasing rate of force redevelopment (K (tr); BA 14.9±1.1s(-1), SHAM 25.2±1.5s(-1)). At the extracellular matrix level, fibrosis (BA 10.8±0.9%, SHAM 5.3±0.6%), pro-MMP-2 and MMP-9 activities increased and, in vivo, relaxation was impaired (τ; BA 14.0±0.9ms, SHAM 12.9±0.4ms). Diabetes increased cardiomyocyte diameter, fibrosis (DM 21.4±0.4μm, 13.9±1.8%, DB 20.6±0.4μm, 13.8±0.8%, respectively), myofilament Ca(2+)sensitivity (DM 5.57±0.02, DB 5.57±0.01), advanced glycation end-product deposition (DM 4.9±0.6score/mm(2), DB 5.1±0.4score/mm(2), SHAM 2.1±0.3score/mm(2)), and apoptosis, while decreasing K (tr) (DM 13.5±1.9s(-1), DB 15.2±1.4s(-1)), Akt phosphorylation and MMP-9/TIMP-1 and MMP-1/TIMP-1 ratios. Diabetic hearts were stiffer (higher end-diastolic-pressure: DM 7.0±1.2mmHg, DB 6.7±0.7mmHg, SHAM 5.3±0.4mmHg, steeper end-diastolic-pressure-volume relation: DM 0.59±0.18, DB 0.83±0.17, SHAM 0.41±0.10), and hypo-contractile (decreased end-systolic-pressure-volume-relation). DB animals presented further pulmonary congestion (Lungs/body-weight: DB 5.23±0.21g/kg, SHAM 3.80±0.14g/kg) as this group combined overload-induced relaxation abnormalities and diabetes-induced stiffness. Diabetes mellitus and pressure overload led to distinct diastolic dysfunction phenotypes: while diabetes promoted myocardial stiffening, pressure overload impaired relaxation. The association of these damages accelerates the progression of diastolic heart failure progression in diabetic-banded animals.