Biomechanical preconditioning of biological specimens by cyclic loading is routinely done presumably to stabilize properties prior to the main phase of a study. However, no prior studies have actually measured these effects for whole bone of any kind. The aim of this study, therefore, was to quantify these effects for whole bones. Fourteen matched pairs of fresh-frozen intact cadaveric canine femurs were sinusoidally loaded in 4-point bending from 50 N to 300 N at 1 Hz for 25 cycles. All femurs were tested in both anteroposterior (AP) and mediolateral (ML) bending planes. Bending stiffness (i.e., slope of the force-vs-displacement curve) and linearity R2 (i.e., coefficient of determination) of each loading cycle were measured and compared statistically to determine the effect of limb side, cycle number, and bending plane. Stiffnesses rose from 809.7 to 867.7 N/mm (AP, left), 847.3 to 915.6 N/mm (AP, right), 829.2 to 892.5 N/mm (AP, combined), 538.7 to 580.4 N/mm (ML, left), 568.9 to 613.8 N/mm (ML, right), and 553.8 to 597.1 N/mm (ML, combined). Linearity R2 rose from 0.96 to 0.99 (AP, left), 0.97 to 0.99 (AP, right), 0.96 to 0.99 (AP, combined), 0.95 to 0.98 (ML, left), 0.94 to 0.98 (ML, right), and 0.95 to 0.98 (ML, combined). Stiffness and linearity R2 versus cycle number were well-described by exponential curves whose values leveled off, respectively, starting at 12 and 5 cycles. For stiffness, there were no statistical differences for left versus right femurs (p = 0.166), but there were effects due to cycle number (p < 0.0001) and AP versus ML bending plane (p < 0.0001). Similarly, for linearity, no statistical differences were noted due to limb side (p = 0.533), but there were effects due to cycle number (p < 0.0001) and AP versus ML bending plane (p = 0.006). A minimum of 12 preconditioning cycles was needed to fully stabilize both the stiffness and linearity of the canine femurs. This is the first study to measure the effects of mechanical preconditioning on whole bones, having some practical implications on research practices.