Human familial hypertrophic cardiomyopathy is the most common Mendelian cardiovascular disease worldwide. Among the most severe presentations of the disease are those in families heterozygous for the mutation R403Q in β-cardiac myosin. Mice heterozygous for this mutation in the α-cardiac myosin isoform display typical familial hypertrophic cardiomyopathy pathology. Here, we study cardiomyocytes from heterozygous 403/+ mice. The effects of the R403Q mutation on force-generating capabilities and dynamics of cardiomyocytes were investigated using a dual carbon nanofiber technique to measure single-cell parameters. We demonstrate the Frank-Starling effect at the single cardiomyocyte level by showing that cell stretch causes an increase in amplitude of contraction. Mutant 403/+ cardiomyocytes exhibit higher end-diastolic and end-systolic stiffness than +/+ cardiomyocytes, whereas active force generation capabilities remain unchanged. Additionally, 403/+ cardiomyocytes show slowed relaxation dynamics. These phenotypes are consistent with increased end-diastolic and end-systolic chamber elastance, as well as diastolic dysfunction seen at the level of the whole heart. Our results show that these functional effects of the R403Q mutation are cell-intrinsic, a property that may be a general phenomenon in familial hypertrophic cardiomyopathy.