Dynamic mechanical loads have been known to stimulate bone formation. Many biophysical factors such as number of daily loading cycles, bone strain, strain-induced interstitial fluid flow, molecular transport, and modulation of intramedullary pressure have been considered as potential mediators in mechanotransduction of bone. Using a knee loading modality that enhances anabolic responses in mouse hindlimb, we addressed a question: Do oscillatory loads applied to the knee induce dynamic alteration of intramedullary pressure in the femoral medullary cavity? To answer this question, mechanical loads were applied to the knee with a custom-made piezoelectric loader and intramedullary pressure in the femoral medullary cavity was measured with a fiber optic pressure sensor. We observed that in response to sinusoidal forces of 0.5 Hz and 10 Hz, pressure amplitude increased up to 4-N loads and reached a plateau at 130 Pa. This amplitude significantly decreased with a loading frequency above 20 Hz. To confirm alteration of intramedullary pressure, real-time motion of microparticles in a glass tube inserted to the femoral medullary cavity ex vivo was visualized. Taken together, these data reveal that knee loading dynamically alters intramedullary pressure as a function of loading intensities and frequencies.