In this paper lubricant film thickness for bovine serum (BS) was measured in a ball-on-disc optical device under steady-state rolling and sliding. Tests were carried out for a range of BS concentrations and substrate materials (M52100 steel and chromium coatings) in both low (MPa) and high-pressure (GPa) configurations. The results show that BS forms films 2–50 nm thick over the speed range although this depends on the contact pressure. However there was significant scatter in these results, possibly due to the inherent nature of the fluid, which is an inhomogeneous biological sample. Clearly this will contribute to scatter in wear results. In some cases thick (up to 100nm) films were formed at low speeds under both sliding and rolling conditions, this behaviour was considered representative of high-viscosity surface layers rather than solid films. However residual films of 13–17nm were also measured under static loading. These are attributed to the adsorption of protein molecules and will provide surface protection during stance or on initiation of gait. A small number of results at under low pressure sliding conditions indicated that much thicker films were formed than at high pressures. One interesting aspect of the results is that they are not representative of a simple Newtonian fluid and thus have considerable implications for the development of predictive film thickness models.