Extended periods of inactivity cause severe bone loss and concomitant deterioration of the musculoskeletal system. Considerable research has been aimed at better understanding the mechanisms and consequences of bone loss due to unloading and the associated effects on strength and fracture risk. One factor that has not been studied extensively but is of great interest, particularly for human spaceflight, is how multiple or repeated exposures to unloading and reloading affect the skeleton. Space agencies worldwide anticipate increased usage of repeat-flier crewmembers, and major thrust of research has focused on better understanding of microgravity effects on loss of bone density at weightbearing skeletal sites; however there is limited data available on repeat microgravity exposure. The adult hindlimb unloaded (HU) rat model was used to determine how an initial unloading cycle will affect a subsequent exposure to disuse and recovery thereafter. Animals underwent 28 days of HU starting at 6 months of age followed by 56 days of recovery, and then another 28 days of HU with 56 days of recovery. In vivo longitudinal pQCT was used to quantify bone morphological changes, and ex vivo μCT was used to quantify trabecular microarchitecture and cortical shell geometry at the proximal tibia metaphysis (PTM). The mechanical properties of trabecular bone were examined by the reduced platen compression mechanical test. The hypothesis that the initial HU exposure will mitigate decrements in bone mass and density for the second HU exposure was supported as pre- to post-HU declines in total BMC, total vBMD, and cortical area by in vivo pQCT at the proximal tibia metaphysis were milder for the second HU (and not significant) compared to an age-matched single HU (3% vs. 6%, 2% vs. 6%, and 2% vs. 6%, respectively). In contrast, the hypothesis was not supported at the microarchitectural level as losses in BV/TV and Tb.Th. were similar during 2nd HU exposure and age-matched single HU. Recovery with respect to post-HU values and compared to aging controls for total BMC, vBMD and cortical area were slower in older animals exposed to single or double HU cycles compared to recovery of younger animals exposed to a single HU bout. Despite milder recovery at the older age, there was no difference between unloaded animals and controls at the end of second recovery period. Therefore, the data did not support the hypothesis that two cycles of HU exposure with recovery would have a net negative effect. Mechanical properties of trabecular bone were affected more severely than densitometric measures (35% loss in trabecular bone ultimate stress vs. 9% loss in trabecular vBMD), which can be attributed most prominently to reductions in trabecular bone density and tissue mineral density. Together, our data demonstrate that initial exposure to mechanical unloading does not exacerbate bone loss during a subsequent unloading period and two cycles of unloading followed by recovery do not have a cumulative net negative effect on total bone mineral content and density as measured by pQCT at the proximal tibia metaphysis.