Vertebral fractures are the most common osteoporotic fractures, but clinical means for assessment of vertebral bone integrity are limited in accuracy, as they typically use surrogate measures that are indirectly related to mechanics. The objective of this study was to examine the extent to which intravertebral strain distributions and changes in cancellous bone texture generated by a load of physiological magnitude can be characterized using a clinically available imaging modality. We hypothesized that digital tomosynthesis-based digital volume correlation (DTS-DVC) and image texture-based metrics of cancellous bone microstructure can detect development of mechanical strains under load. Isolated cadaveric T11 vertebrae and L2–L4 vertebral segments were DTS imaged in a nonloaded state and under physiological load levels. Axial strain, maximum principal strain, maximum compressive and tensile principal strains, and von Mises equivalent strain were calculated using the DVC technique. The change in textural parameters (line fraction deviation, anisotropy, and fractal parameters) under load was calculated within the cancellous centrum. The effect of load on measured strains and texture variables was tested using mixed model analysis of variance, and relationships of strain and texture variables with donor age, bone density parameters, and bone size were examined using regression models. Magnitudes and heterogeneity of intravertebral strain measures correlated with applied loading and were significantly different from background noise. Image texture parameters were found to change with applied loading, but these changes were not observed in the second experiment testing L2–L4 segments. DTS-DVC-derived strains correlated with age more strongly than did bone mineral density (BMD) for T11.