Using a microfluidic platform to apply negative aspiration pressure (-20, -25, -30, -35 and -40 cm H2O), we compared the differences in creep responses of Glioblastoma Multiforme (GBM) cells while migrating in confinement and at a stationary state on a 2D substrate. Cells were either migrating in a channel of 5 x 5 μm cross-section or stationary at the entrance to the channel. In response to aspiration pressure, we found actively migrating GBM cells exhibited a higher stiffness than stationary cells. Additionally, migrating cells absorbed more energy elastically with a relatively small dissipative energy loss. At elevated negative pressure loads up to - 30 cm H2O, we observed a linear increase in elastic deformation and a higher distribution in elastic storage than energy loss, and the response plateaued at further increasing negative pressure loads. To explore the underlying cause, we carried out immuno-cytochemical studies of these cells and found a polarized actin and myosin distribution at the front and posterior ends of the migrating cells, whereas the distribution of the stationary group demonstrated no specific regional differences. These differences in creep response and cytoskeletal protein distribution demonstrate the importance of a migrating cell's kinematic state to the mechanism of cell migration.