Muscle stem cells (MuSCs) are requisite for skeletal muscle regeneration and homeostasis. Proper functioning of MuSCs, including activation, proliferation, and fate decision, is determined by an orchestrated series of events and communication between MuSCs and their niche. A multitude of biochemical stimuli are known to regulate MuSC fate and function. However, in addition to biochemical factors, it is conceivable that MuSCs are subjected to mechanical forces during muscle stretchshortening cycles because of myofascial connections between MuSCs and myofibers. MuSCs respond to mechanical forces in vitro, but it remains to be proven whether physical forces are also exerted on MuSCs in their native niche and whether they contribute to the functioning and fate of MuSCs. MuSC deformation in their native niche resulting from mechanical loading of ex vivo myofiber bundles was visualized utilizing mT/mG double-fluorescent Cre-reporter mouse and multiphoton microscopy. MuSCs were subjected to 1 h pulsating fluid shear stress (PFSS) with a peak shear stress rate of 6.5 Pa/s. After PFSS treatment, nitric oxide, messenger RNA (mRNA) expression levels of genes involved in regulation of MuSC proliferation and differentiation, ERK 1/2, p38, and AKT activation were determined. Ex vivo stretching of extensor digitorum longus and soleus myofiber bundles caused compression as well as tensile and shear deformation of MuSCs in their niche. MuSCs responded to PFSS in vitro with increased nitric oxide production and an upward trend in iNOS mRNA levels. PFSS enhanced gene expression of c-Fos, Cdk4, and IL-6, whereas expression of Wnt1, MyoD, Myog, Wnt5a, COX2, Rspo1, Vangl2, Wnt10b, and MGF remained unchanged. ERK 1/2 and p38 MAPK signaling were also upregulated after PFSS treatment. We conclude that MuSCs in their native niche are subjected to force-induced deformations due to myofiber stretch-shortening. Moreover, MuSCs are mechanoresponsive, as evidenced by PFSS-mediated expression of factors by MuSCs known to promote proliferation.