ABSTRACTScanning acoustic microscopy (SAM), when applied to biological samples has the potential to resolve the longitudinal acoustic wave speed and hence stiffness of discrete tissue components. The heterogeneity of biological materials combined with the action of cryosectioning and rehydrating can, however, create variations in section topography. Here, we set out to determine how variations in specimen thickness influence apparent acoustic wave speed measurementsCryosections (5μm nominal thickness) of human skin biopsies were adhered to glass slides before washing and rehydrating in water. Multiple regions (200x200 μm; n = 3) were imaged by SAM to generate acoustic wave speed maps. Subsequently co-localised 30x30 μm sub-regions were imaged by atomic force microscopy (AFM) in fluid. The images were then registered using Image J. Each pixel was allocated both a height and wave speed value before their relationship was then plotted on a scattergram. The mean section thickness measured by AFM was 3.48 ± 1.12 (SD) μm. Regional height variations influenced apparent wave speed measurements. A 3.5 μm height difference was associated with a 400 ms^-1 increase in wave speed. In the present study we show that local variations in specimen thickness influence apparent wave speed. We also show that a true measure of wave speed can be calculated if the thickness of the specimen is known at each sampling point.