Objective: Diastolic heart failure (DHF) is characterized, e.g., by delayed relaxation, impaired left ventricular (LV) filling and increased LV stiffness. Titin is an established contributor to LV stiffness, but little is known about the protein's contribution to altered diastolic function in DHF. Here we studied whether cardiac titin-isoform switching contributes to increased passive stiffness in DHF. N2B-titin contains a stiffer, N2BA-titin a more compliant spring. Methods: We investigated LV tissue samples of two animal models of DHF and interventricular septum samples of aortic stenosis (AS) patients, for titin-isoform composition by loose-gel electrophoresis and titin-based stiffness by skinned-fiber mechanics. Results: Induction of diastolic dysfunction in the "two kidney one clip" (2K1C) rat model, which develops LV hypertrophy due to chronic afterload increase, caused no significant changes in the titin-isoform expression pattern, both 6 weeks and 8 months following surgery (~6% N2BA in both 2K1C and SHAM-operated LV, the remainder being N2B isoform). In contrast, in an old dog DHF model (8-12 years) made hypertensive by bilateral renal wrapping, the cardiac N2BA proportion was significantly lower (35.5±5.1%; mean±SD) than in normal old dog LV (43.1±4.8%; p<0.01). Mechanical measurements revealed passive- stiffness modulations consistent with the magnitude of titin-isoform switching. In contrast, in human AS samples the N2BA titin proportion was 42.0±4.0%, slightly higher than in location-matched normal donor hearts (37.5±5.0%; p<0.025). Conclusion: We conclude that titin-isoform switching is associated with diastolic dysfunction in a large animal model and in humans. Stiffer titin isoforms are expressed in the patient-mimicking dog model of DHF, whereas the small shift towards more compliant titin in aortic stenosis patients may be unrelated to a DHF phenotype. Titin stiffening due to isoform switching may contribute to increased diastolic stiffness in DHF.