We investigated the effects of hindlimb unloading (HLU) on malondialdehyde (MDA), a biomarker for oxidative stress, and glutathione (GSH) levels in tissues of rats. Aminoguanidine (AG), a nucleophilic hydralazine compound and an in vivo antioxidant against reactive oxygen species (ROS) and lipid peroxidation, was used to confirm the HLU-induced oxidative response. Three groups of rats were used: Group 1 was a loaded control group that was maintained on drinking water only; Groups 2 and 3 were hindlimb unloaded (HLU) groups that were maintained on drinking water and on AG in drinking water, respectively. The hindlimb unloaded rats maintained on tap water had significantly elevated MDA levels in 7 tissues (brain, lung, pancreas, kidney, intestine, heart, liver) when compared to the paired hindlimb loaded controls (p <0.05). In contrast, the hindlimb unloaded rats maintained on AG in drinking water had no increase in tissue MDA levels when compared to the loaded controls; moreover, their tissue MDA levels were significantly reduced from the HLU group on tap water (p <0.05). In HLU rats maintained on AG, there were no changes in tissue GSH levels with the exception of brain, where GSH levels were significantly reduced when compared to the other groups (p <0.05). In summary, HLU induced an oxidative response in rats and this response was reduced significantly by ingestion of AG. These results suggest the potential application of AG in the diet of astronauts living in a stressful environment.