SummaryCells residing in the cardiac niche are constantly experiencing physical stimuli, including electrical pulses and cyclic mechanical stretch. These physical signals are known to influence a variety of cellular functions, including the secretion of growth factors and extracellular matrix proteins in cardiac fibroblasts, calcium handling and contractility in cardiomyocytes, or stretch-activated ion channels in muscle and non-muscle cells of the cardiovascular system. Recent progress in cardiac tissue engineering suggests that controlled physical stimulation can lead to functional improvements in multicellular cardiac tissue constructs. To study these effects, aspects of the physical environment of the myocardium have to be mimicked in vitro. This protocol demonstrates how a specifically designed bioreactor system allows controlled exposure of cultured cells, during continuous live imaging, to cyclic stretch, rhythmic electrical stimulation, fluid perfusion, at user-defined temperatures.