Abstract Body: Diastolic dysfunction is present in many patients with heart failure with preserved ejection fraction (HFpEF); however the basis for impaired diastole at the myofilament level remains unresolved. We previously reported that cardiomyocytes (CMs) from patients with a ‘classic’ HFpEF subphenotype, dominated by hypertension and hypertrophy (HH), had elevated, fibrosis-independent resting stiffness and preserved force development at systolic Ca²⁺ levels compared to non-failing controls (NF). CMs from patients with morbid obesity/diabetes (OD), or from those displaying a combination of phenotypes (Mixed), showed depressed maximal tension at systolic Ca²⁺ vs NF. We also observed excessive diastolic tension in HH and Mixed CMs, suggesting distinct mechanisms may impede myofilament relaxation to elevate diastolic while preserving systolic force. Myofilament relaxation, however, cannot be measured in CMs. We hypothesized that CM-level deficiencies stem from compromised myofilament function and that, minimally, myofibrils from HH and Mixed biopsies would exhibit impaired relaxation. Therefore, we investigated activation/relaxation kinetics and mechanics in myofibrils isolated from HH (n=68), OD (n=60), Mixed (n=63), and NF (n=67) endocardial biopsies (5 patients/group), using a custom-built apparatus. The population was 55% female, 55% White, 25% Black, and 59±8 yrs. Compared to NF (51.61±1.97 kPa), average maximal Ca²⁺-activated tension was lower in OD (39.8±1.6 kPa, p<0.0001) and Mixed (41.2±1.4 kPa, p=0.0003), but not in HH myofibrils (46.8±2.0 kPa, p=0.2103), reminiscent of CM data. Upon Ca²⁺ removal, initial linear relaxation was prolonged in all HFpEF groups (in msec: HH=247±5, p<0.0001; OD=215±5, p=0.0421; Mixed=249±6, p<0.0001) vs NF (193±6), indicating excessive actomyosin interactions due to delayed myofilament inactivation. In conclusion, we identified a primary myofibrillar defect in maximal tension in myocardium of highly obese HFpEF patients, and impaired fibrillar relaxation in all subgroups, which could significantly contribute to the observed organ-level diastolic dysfunction. Treatments targeting myofilament dysfunction may prove beneficial in this syndrome, but the degree of obesity should be considered.