Contractile function is primarily determined by the myosin heavy chain (MyHC). The β-isoform is the most often expressed in human ventricular cardiomyocytes (CMs). We previously showed that following long-term incubation on glass coverslips covered with laminin, approximately 80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express just β-MyHC. In order to characterise cellular function, we examined the effects of enzymatically separating hESC-CMs following prolonged growth and then replating them. In a micro-mechanical setting, we found that force-related kinetic characteristics reflected variations in calcium transients and resembled α- rather than β-MyHC-expressing myofibrils. According to single-cell immunofluorescence analysis, replating hESC-CMs caused α-MyHC to be rapidly upregulated, as evidenced by increases in hESC-CMs that expressed α-MyHC solely and α/β-MyHC mixed. Individual CMs generated from human induced pluripotent stem cells (hiPSCs) similarly showed a similar increase in the variability of MyHC isoform expression following replating. During the second week following replating, the alterations in cardiomyocyte function and MyHC isoform expression brought on by replating were reversible. The expression profile of genes and pathways relevant to mechanosensation/transduction, particularly integrin-associated signaling, changed, according to gene enrichment analysis based on RNA-sequencing data. Thus, focal adhesion kinase (FAK), an integrin downstream mediator, enhanced β-MyHC expression on a rigid matrix, thereby confirming gene enrichment analysis. In conclusion, long-term cultivated human stem cell-derived CMs underwent significant changes in gene expression, MyHC isoform composition, and function as a result of detachment and replating. This led to changes in mechanosensation/transduction, which should be taken into account, especially for subsequent in vitro tests.