This study aimed to compare alterations in phosphorylation and function of sarcomeric proteins in diastolic heart failure (DHF) vs. normal myocardium under two conditions of tissue procurement: beating-heart biopsy and postmortem tissue sampling. Left ventricular tissue samples were procured from normal (CTRL) or old dogs made hypertensive by renal wrapping (DHF), by either taking biopsies of the beating heart (n=7) or excising tissue postmortem (n=8). Isolated permeabilized cardiomyocytes were attached to a force transducer and passive tension (Fpassive) was measured between 1.8 and 2.4 μm, calcium sensitivity (pCa50) at 2.2 μm sarcomere length. Phosphorylation of myofilament proteins was assessed using the SYPRO-Ruby (total protein) / Pro-Q Diamond (phosphoprotein) system. Expression of total or phosphorylated protein, including titin-PEVK, cTnI, and PKCα was also quantified by immunoblot. Postmortem tissues and biopsy samples showed similar changes in DHF vs. CTRL, including increased phospho-PEVK, phospho-PKCα, and Fpassive, but decreased titin N2BA:N2B isoform ratio and reduced phosphorylation of total titin, cMyBPC, cTnT, cTnI and cTnI (S23/24). Differences were apparent in terms of lowered pCa50 in postmortem DHF, but increased pCa50 in biopsy DHF. Reduced MLC2 phosphorylation in DHF vs. CTRL was observed in biopsies, but not in postmortem tissues. A degradation form of titin (T2) was more abundant in postmortem tissues than in biopsies. We conclude that this DHF model is characterized by titin-isoform shift towards the stiff N2B isoform, a deficit in total titin phosphorylation, but increases in PKCα-dependent phosphorylation of titin-PEVK-domain and cardiomyocyte Fpassive, as well as altered pCa50. For a few parameters, postmortem and beating-heart samples differed in the direction of change in DHF vs. CTRL, particularly regarding Ca2+-sensitivity. However, most parameters changed in the same direction, suggesting the cellular events associated with death alter sample properties to a modest degree.