Many cardiac therapeutic modalities, including pacemakers, implantable cardioverter defibrillators, and cardiac resynchronization therapy devices, are used to treat abnormalities in cardiac function and conduction. Both electrical and mechanical dyssynchrony can have deleterious effects including reduced cardiac output and an increased susceptibility to cardiac arrhythmias. It is postulated that electromechanical dyssynchrony may contribute to the susceptibility of the heart to cardiac arrhythmias. In this study, a novel system was developed to study these effects by altering the electro-mechanical activation sequence in cultured neonatal rat cardiomyocyte monolayers by dyssynchronously stimulating the monolayers with applied electrical fields and pulsatile mechanical strain. Specifically, optical mapping was utilized to compare action potential duration and quantify arrhythmia susceptibility of cardiomyocytes subjected to pulsatile mechanical strain, electrical stimulation, and dyssynchronous electrical and mechanical stimulation. This system provides a method to evaluate changes in cardiomyocyte conduction properties due to altered electro-mechanical coupling and the subsequent impact on arrhythmogenesis.