Neutrophils traversing the pulmonary microcirculation are subjected to mechanical stimulation during their deformation into narrow capillaries. To better understand the time-dependant changes caused by this mechanical stimulus, neutrophils were caused to flow into a microchannel, which allowed simultaneous visualization of cell morphology and passive rheological measurement by tracking the Brownian motion of endogenous granules. Above a threshold stimulus, mechanical deformation resulted in neutrophil activation with pseudopod projection. The activation time was inversely correlated to the rate of mechanical deformation experienced by the neutrophils. A reduction in shear moduli was observed within seconds after the onset of the mechanical stimulus, suggesting a sudden disruption of the neutrophil cytoskeleton when subjected to mechanical deformation. However, the magnitude of the reduction in moduli was independent of the degree of deformation. Recovery to nearly the initial values of viscoelastic moduli occurred within 1 min. These observations confirm that mechanical deformation of neutrophils, similar to conditions encountered in the pulmonary capillaries, is not a passive event; rather, it is capable of activating the neutrophils and enhancing their migratory tendencies.