Background— To support the clinical distinction between systolic heart failure (SHF) and diastolic heart failure (DHF), left ventricular (LV) myocardial structure and function were compared in LV endomyocardial biopsy samples of patients with systolic and diastolic heart failure. Methods and Results— Patients hospitalized for worsening heart failure were classified as having SHF (n=22; LV ejection fraction (EF) 34±2%) or DHF (n=22; LVEF 62±2%). No patient had coronary artery disease or biopsy evidence of infiltrative or inflammatory myocardial disease. More DHF patients had a history of arterial hypertension and were obese. Biopsy samples were analyzed with histomorphometry and electron microscopy. Single cardiomyocytes were isolated from the samples, stretched to a sarcomere length of 2.2 μm to measure passive force (F passive ), and activated with calcium-containing solutions to measure total force. Cardiomyocyte diameter was higher in DHF (20.3±0.6 versus 15.1±0.4 μm, P <0.001), but collagen volume fraction was equally elevated. Myofibrillar density was lower in SHF (36±2% versus 46±2%, P <0.001). Cardiomyocytes of DHF patients had higher F passive (7.1±0.6 versus 5.3±0.3 kN/m 2 ; P <0.01), but their total force was comparable. After administration of protein kinase A to the cardiomyocytes, the drop in F passive was larger ( P <0.01) in DHF than in SHF. Conclusions— LV myocardial structure and function differ in SHF and DHF because of distinct cardiomyocyte abnormalities. These findings support the clinical separation of heart failure patients into SHF and DHF phenotypes.