Abstract Body (Do not enter title and authors here): Heart failure with preserved ejection fraction (HFpEF) has long been managed with pharmacologic heart rate reduction, in part to increase ventricular filling time. However, this approach overlooks the increased end-diastolic pressure-volume relationship (i.e., stiffness) of HFpEF myocardium, which produces a disproportionate rise in filling pressure at reduced heart rates. Considering that emerging clinical data show symptomatic benefit from accelerated pacing in HFpEF patients, we tested the hypothesis that accelerated pacing enhances myocardial performance by reducing passive stiffness in a rodent model of diastolic dysfunction. Left ventricular myocardial slices were isolated from ZSF1-lean and -obese rat myocardium. Slices were subjected to baseline (1 Hz) or accelerated (2 Hz) pacing, and force-length work loops were recorded. Accelerated pacing for two hours decreased the end-diastolic force-length relationship (34.9 ± 6.6 vs. 6.9 ± 1.4 mN/mm, p=0.0159) in ZSF1-lean and -obese myocardial slices, indicating a significant reduction in ventricular stiffness. As a functional consequence of decreased stiffness, pacing reduced isovolumic contraction (IVC; 41.5 ± 0.2 vs. 27.2 ± 0.3 ms, p=0.0159) and relaxation times (IVR; 50.8 ± 7.6 vs. 18.8 ± 7.9 ms, p=0.0635) while increasing the duration of ejection (86.8 ± 4.1 vs. 121.4 ± 5.6 ms, p=0.0159). Interestingly, cardiac slices treated with accelerated pacing also exhibited a greater stroke length per unit of force (7.8 ± 0.2 vs. 20.3 ± 0.1 mm/mN, p=0.0159), demonstrating an increased efficiency likely driven by the reduction in stiffness. Given that these stiffness-driven changes have considerable implications for HFpEF patients, cardiac slices from ZSF1-obese rats were subjected to continuous pacing overnight, mimicking current clinical trials, and their passive mechanical properties were defined. Application of a rapid stretch (20% diastolic length in 100 ms) revealed a decreased peak elastic modulus (11.6 ± 4.2 vs. 3.4 ± 1.9 mN/mm2, p=0.0028) and steady state stress (4.0 ± 1.0 vs. 2.0 ± 1.2 mN/mm2, p=0.0050), marking the first demonstration that accelerated pacing can reverse pathological ventricular stiffening in diastolic dysfunction. Taken together, accelerated pacing decreases the end-diastolic force-length relationship, IVC, IVR, and increases efficiency in rodent HFpEF myocardium, which may underlie clinically-observed benefits in exercise tolerance, atrial fibrillation burden, and quality of life.