The factors regulating lung fluid balance during inflammation are not completely understood. The glycocalyx of lung endothelial cells is believed to play a significant role in permeability via the transduction of extracelluar mechanical stimuli into intracellular chemical signals. Specifically, heparan sulfates and hyaluronan were proposed to have control over the biomechanical properties of the glycocalyx. Here we used a combination of particle probe reflectance interference contrast microscopy (RICM) and atomic force microscopy (AFM) to characterize the in vitro biomechanical properties of major components of the glycocalyx in bovine lung microvascular endothelial cells (BLMVEC). In RICM experiments, spherical glass beads were randomly deposited on a confluent monolayer of BLMVECs. By recording the bead interference fringe pattern in cell-cell junction regions as a function of time, a particle’s vertical position and its fluctuations around the equilibrium position were determined. In AFM experiments, glass bead was glued to a soft AFM cantilever and a simple, quasi-static indentation of the endothelial cells was recorded as a function of loading force. The RICM/AFM results were acquired before and after selective enzymatic digestion of major glycocalyx components. The effective spring constants were elucidated from the bead energy-height profiles produced by the thermal fluctuations of the beads (RICM) and from AFM force-indentation data.