This work introduces NeoMag, a system designed to enhance cell mechanics assays in substrate deformation studies. NeoMag uses multidomain magneto-active materials to mechanically actuate the substrate, transmitting reversible mechanical cues to cells. The system boasts full flexibility in alternating loading substrate deformation modes, seamlessly adapting to both upright and inverted microscopes. The multidomain substrates facilitate mechanobiology assays on 2D and 3D cultures. The integration of the system with nanoindenters allows for precise evaluation of cellular mechanical properties under varying substrate deformation modes. The system is used to study the impact of substrate deformation on astrocytes, simulating mechanical conditions akin to traumatic brain injury and ischemic stroke. The results reveal local heterogeneous changes in astrocyte stiffness, influenced by the orientation of subcellular regions relative to substrate strain. These stiffness variations, exceeding 50% in stiffening and softening, and local deformations significantly alter calcium dynamics. Furthermore, sustained deformations induce actin network reorganization and activate Piezo1 channels, leading to an initial increase followed by a long-term inhibition of calcium events. Conversely, fast and dynamic deformations transiently activate Piezo1 channels and disrupt the actin network, causing long-term cell softening. These findings unveil mechanical and functional alterations in astrocytes during substrate deformation, illustrating the multiple opportunities this technology offers., C. G.-C. and M. F.-d.l.T. authors contributed equally to this work. The authors thank Lotte van Dasler for the relevant discussion. The authors acknowledged support from the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Program (Grant agreement no. 947723, project: 4D-BIOMAP and Grant agreement no. 101081713, project: ISBIOMECH). G.P. acknowledged support from PID2019-106579RB-I00 funded by MCIN/AEI/10.13039/5011 00011033 and by “ERDF A way of making Europe”, and La Marató TV3 Foundation #225619. AMB acknowledged support from Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación Grant PID2019-109820RB-I00, MCIN/AEI/10.13039/501100011033/, co-financed by the European Regional Development Fund (ERDF), ‘A way of making Europe’. C.G.-C. acknowledged support from the Ministerio de Universidades, Spain (FPU20/01459) and A.M.B. from Universidad Carlos III de Madrid ('Ayudas para la recualificación del profesorado funcionario o contratado')., Peer reviewed