Our body’s functioning depends on the ability of cells to sense and react to their local mechanical environment; this process is known as mechanotransduction. Despite the importance of understanding how cells interact with mechanical stimuli, the specific mechanisms governing such processes have yet to be elucidated. Using microscopy to detect the early responses of living cells to mechanical loads and forces would be a critical step towards further understanding cellular mechanotransduction. Dynamic and high-frequency cyclical loads are relevant to human physiology and disease. Yet, modern microscopy systems are not capable of delivering the appropriate mechanical stimuli to live cell cultures. To address this deficiency, we developed a suite of mechanostimulation platforms that provide precise and relevant loads and forces to cell cultures during simultaneous microscopic analysis. We developed a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Using this system, we found that vibration of osteoblastic cells does not elicit acute elevation of cytosolic free calcium, but did desensitize responses to later stimulation with extracellular ATP. We next developed and validated a technique for the practical fabrication of microfluidic channels. In contrast to the effect of vibration, osteoblastic cells were found to respond to changes in fluid shear stress with transient elevation in the concentration of cytosolic free calcium. Lastly, we developed a system to apply disturbed fluid flow to live cells during real-time imaging. This system was used to demonstrate changes in the concentration of cytosolic free calcium in human endothelial cells exposed to laminar and disturbed flow. Our findings indicate that different forms of mechanical stimuli activate distinct signaling pathways in cells. Moreover, these new technologies will facilitate investigations of the signaling pathways activated by dynamic mechanical stimulation of a variety of cell types, in particular those of the skeletal and vascular systems
