The tissues of hollow organs can routinely stretch up to 2.5 times their length. Although significant pathology can arise if relatively large stretches are sustained, the responses of cells are not known at these levels of sustained strain. A key challenge is presenting cells with a realistic and well-defined three-dimensional (3D) culture environment that can sustain such strains. Here, we describe an in vitro system called microscale, magnetically-actuated synthetic tissues (micro-MASTs) to quantify these responses for cells within a 3D hydrogel matrix. Cellular strain-threshold and saturation behaviors were observed in hydrogel matrix, including strain-dependent proliferation, spreading, polarization, and differentiation, and matrix adhesion retained at strains sufficient for apoptosis. More broadly, the system shows promise for defining and controlling the effects of mechanical environment upon a broad range of cells.