In approximately half of heart failure patients, systolic function is near normal while diastolic function is impaired. This condition is therefore referred to as diastolic heart failure (DHF) or heart failure with preserved ejection fraction (HFpEF). The mechanisms underlying the diastolic dysfunction are still unclear, but may be attributed to stiffening of the myocardium. Alterations in the extracellular matrix and cytoskeleton have been reported to contribute to greater passive stiffness in this condition (1). However, the contribution of altered Ca2+ handling to the active process of cardiomyocyte relaxation remains largely uninvestigated. Hypertrophy due to aortic stenosis is one of several etiologies associated with DHF, and with aortic banding (AB) we similarly induced hypertrophy and diastolic dysfunction in Wistar rats. All procedures were performed during inhalation anesthesia (65% N2O, 32% O2, 2.5% isoflurane). Experiments were performed 6 weeks after aortic banding. Rats with detectable systolic heart failure (reduced left ventricular shortening) were excluded. Compared to sham-operated controls, echocardiography revealed hypertrophy and diastolic dysfunction in AB. This was indicated by increased posterior wall thickness and decreased peak early diastolic tissue velocity. The in vivo diastolic dysfunction was confirmed by isometric force measurements in myocardial strips. We observed longer time to peak force and slower relaxation in AB across a range of stimulation frequencies (0.5-6 Hz). Surprisingly, isolated cardiomyocytes exhibited the opposite features. Single cell contractions, measured by edge detection, revealed significantly faster time to peak contraction and faster relaxation in AB. At high frequencies, AB cardiomyocytes also showed less diastolic contracture than sham cells. The mechanism underlying the improved single cell relaxation appeared to be faster cytoplasmic Ca2+ removal, evident as more rapid decay of Ca2+ transients (whole-cell fluorescence, fluo 4-AM) in AB across a range of frequencies. Furthermore, with increasing pacing rate AB cardiomyocytes exhibited a less pronounced increase in diastolic [Ca2+] than sham, and elevated SR Ca2+ content. These observations were consistent with a measured increase in the rates of both sarcoplasmic reticulum Ca2+ reuptake and Ca2+ extrusion in AB. Confocal imaging of cells stained with di-8-ANEPPs indicated maintained t-tubule organization, consistent with an unaltered time to peak of Ca2+ transients. Our results consequently indicate that despite improved Ca2+ handling, AB induces diastolic dysfunction in vivo as well as in intact myocardium ex vivo. We therefore suggest that increased passive stiffness is the major contributor to the impaired diastolic function in this condition.