The vertebral strength and strain can be assessed in vitro by both using isolated vertebrae and sets of three adjacent vertebrae (the central one is loaded through the disks). Our goal was to elucidate if testing single-vertebra-specimens in the elastic regime provides different surface strains to three-vertebrae-segments. Twelve three-vertebrae sets were extracted from thoracolumbar human spines. To measure the principal strains, the central vertebra of each segment was prepared with eight strain-gauges. The sets were tested mechanically, allowing comparison of the surface strains between the two boundary conditions: first when the same vertebra was loaded through the disks (three-vertebrae-segment) and then with the endplates embedded in cement (single-vertebra). They were all subjected to four nondestructive tests (compression, traction, torsion clockwise, and counterclockwise). The magnitude of principal strains differed significantly between the two boundary conditions. For axial loading, the largest principal strains (along vertebral axis) were significantly higher when the same vertebra was tested isolated compared to the three-vertebrae-segment. Conversely, circumferential strains decreased significantly in the single vertebrae compared to the three-vertebrae-segment, with some variations exceeding 100% of the strain magnitude, including changes from tension to compression. For torsion, the differences between boundary conditions were smaller. This study shows that, in the elastic regime, when the vertebra is loaded through a cement pot, the surface strains differ from when it is loaded through the disks. Therefore, when single vertebrae are tested, surface strain should be taken with caution.