Environmental stiffness is a crucial determinant of cell function. There is a long-standing quest for reproducible and (human matrix) bio-mimicking biomaterials with controllable mechanical properties to unravel the relationship between stiffness and cell behavior. Here, we evaluate methacrylated human recombinant collagen peptide (RCPhC1-MA) hydrogels as a matrix to control 3D microenvironmental stiffness and monitor cardiac cell response. We show that RCPhC1-MA can form hydrogels with reproducible stiffness in the range of human developmental and adult myocardium. Cardiomyocytes (hPSC-CMs) and cardiac fibroblasts (cFBs) remain viable for up to 14days inside RCPhC1-MA hydrogels while the effect of hydrogel stiffness on extracellular matrix production and hPSC-CM contractility can be monitored in real-time. Interestingly, whereas the beating behavior of the hPSC-CM monocultures is affected by environmental stiffness, this effect ceases when cFBs are present. Together, we demonstrate RCPhC1-MA to be a promising candidate to mimic and control the 3D biomechanical environment of cardiac cells.