Disc hydration is controlled by fluid imbibition and exudation and hence by applied load magnitude and history, internal osmotic pressure and disc conditions. It affects both the internal load distribution and external load-bearing of a disc while variations therein give rise to the disc time-dependent characteristics. This study aimed to evaluate the effect of changes in compression preload magnitude on the disc axial cyclic compression stiffness under physiological loading. After 20h of free hydration, effects of various preload magnitudes (no preload, 0.06 and 0.28MPa, applied for eight hours) and disc-bone preparation conditions on disc height and axial stiffness were investigated using 36 disc-bone and 24 isolated disc (without bony endplates) bovine specimens. After preloading, specimens were subjected to ten loading/unloading cycles each of 7.5min compression at 0.5MPa followed by 7.5min at 0.06MPa. Under 0.06MPa preload, the specimen height losses during high loading periods of cyclic loading were greater than corresponding height recoveries during low loading phases. This resulted in a progressive reduction in the specimen height and increase in its stiffness. Differences between disc height losses in high cyclic loads and between stiffness in both load increase and release phases were significant for 0 and 0.06MPa vs. 0.28MPa preload. Results highlight the significant role of disc preload magnitude/history and hence disc height and hydration on disc stiffness in loading/unloading and disc height loss in loading periods. Proper preconditioning and hence hydration level should be achieved if recovery in height loss similar to in vivo conditions is expected.