Functional spinal units (FSUs) are frequently used for in vitro mechanical testing. This approach assumes that the mechanical behavior of the FSUs is equivalent to the mechanics of these segments within the intact spine. The purpose of this study was to determine whether normal spinal mechanics are compromised in FSU preparations. Flexion-extension pure-moment flexibility testing was performed on 13 L2-L5 porcine specimens. The moment-angle relationship of the L3/L4 segment was recorded, and then the multisegment specimens were cut down to L3/L4 FSUs and retested. Comparisons of stiffness, range of motion, and laxity zone were made between conditions. The neutral zone and range of motion parameters were significantly larger in the L3/L4 motion segment compared to the L3/L4 segment tested within the multisegment specimen; the stiffness was not significantly different. These differences were attributed to cutting the supraspinous ligament as this ligament spans several vertebral levels. Flexion mechanical tests performed on FSUs should be interpreted cautiously as the biomechanics of FSUs is altered from normal. Although the choice of spine length depends on the experimental purpose, spinal flexion studies should be performed on multisegment specimens to appropriately represent the anatomical boundary conditions.