Troponin I (TnI), the inhibitory subunit of the heterotrimeric troponin complex (Tn) is a Ca²⁺- dependent molecular switch that regulates the force generation of the sarcomere and the contraction and relaxation of the heart. We investigated the mechanisms by which mutations on TnI associated to familial hypertrophic cardiomyopathy (FHC) cause diastolic dysfunction, i.e. impair heart filling. Effects of TnI mutations on the molecular level were analyzed by measuring the switch-on and switch-off kinetics of Tn in solution and of Tn in the sarcomere. Furthermore, we characterized their effects on the kinetics of force development and force relaxation. Force was measured using subcellular cardiac myofibrils in which the endogenous Tn was exchanged by recombinant mutant Tn or using myofibrils isolated from transgenic mice that over-express mutant TnI. Finally, the systolic and diastolic heart function of transgenic mice was explored in vivo. FHC-associated mutations in TnI slow down the switch-off of Tn within the sarcomere, cause slower and incomplete myofibrillar force relaxation, finally resulting in a slowed down and incomplete pressure decay of the left ventricle. This diastolic dysfunction manifests without systolic dysfunction, fibrosis, myocyte disarray, ventricular hypertrophy and alterations of Ca²⁺-homeostasis which strengthens the hypothesis that diastolic dysfunction represents an early stage of the disease that rather directly results from impaired molecular interactions of Tn in the sarcomere than secondary from adaptations of the heart. Thus, the diastolic dysfunction in TnI-based cardiomyopathies is the primary cause and not the consequence of the heart disease, and TnI-based cardiomyopathies should be better regarded as a 'primary' diastolic than hypertrophic heart disease.