Presently, there is little consensus about how, or even if, axial preload should be incorporated in spine flexibility tests in order to simulate the compressive loads naturally present in vivo. Some preload application methods are suspected of producing unwanted “artefact” forces as the specimen rotates and, in doing so, influencing the resulting kinematics. The objective of this study was to quantitatively compare four distinct types of preload which have roots in contemporary experimental practice. The specific quantities compared were the reaction moments and forces resulting at the intervertebral disc and specimen kinematics. The preload types incorporated increasing amounts of caudal constraint on the preload application vector ranging from an unconstrained dead-load arrangement to an apparatus that allowed the vector to follow rotations of the specimen. Six human cadaveric spine segments were tested (1-L1/L2, 3-L2/L3, 1-L3/L4 and 1-L4/L5). Pure moments were applied to the specimens with each of the four different types of compressive preload. Kinematic response was measured using an opto-electronic motion analysis system. A six-axis load cell was used to measure reaction forces and moments. Artefact reaction moments and shear forces were significantly affected by preload application method and magnitude. Unconstrained preload methods produced high artefact moments and low artefact shear forces while more constrained methods did the opposite. A mechanical trade-off is suggested by our results, whereby unwanted moment can only be prevented at the cost of shear force production. When comparing spine flexibility studies, caution should be exercised to ensure preload was applied in a similar manner for all studies. Unwanted moments or forces induced as a result of preload application method may render the comparison of two seemingly similar studies inappropriate.