Familial Cardiomyopathy (HCM) is linked to mutations of sarcomeric proteins such as TnI. Mutations of TnI are associated with diastolic dysfunction but it is not known whether this is due to a slowing of the switch-off kinetics of Tn in the myofibril. Hence, we determined the switch-off kinetics of wild type (WT) Tn and 3 TnI-mutants, R21C, R145G and [Delta]K183 located at functionally different sites of TnI. To monitor the kinetics Tn was fluorescently labelled and exchanged for endogenous Tn in guinea pig cardiac myofibrils (cMF). This allows to study the switch kinetics within the structural constrains of the sarcomere. The cMF were Ca2+-activated in a stopped-flow apparatus and then mixed with BAPTA to rapidly lower Ca2+. This caused a exponential fluorescence decay reflecting the conformational change of Tn. For WT-Tn its rate constant was 39±2s-1. All three HCM-associated mutations lead to a slow down of the kinetics (R21C=29±1s-1;R145G=20±4s-1;[Delta]K183=28±3s-1). In addition we have determined the kinetics of force relaxation on exchanged cMF and showed a slowing of the rate constants also for all mutations. Albeit further mutations need to be investigated our study indicates, that the HCM-associated mutations on TnI slow down the switch-off and thereby force relaxation which could be responsible for the diastolic dysfunction.