Aim 1 focused on identifying perturbations in gastrocnemius and quadriceps from mice with hindlimb unloading, space radiation, or a combination.

• Hindlimb unloading led to decreased weight of gastrocnemius muscle; however, the addition of radiation did not have a compounded effect at the time point assessed.

• Untargeted metabolomics at 24h after exposure, with and without hindlimb unloading, identified changes in both gastrocnemius and quadricep muscles, with pathways such as acylcarnitines and tricarboxylic acid cycle, amino acids and derivatives, coenzyme-A metabolism, various cofactors, eicosanoids, fatty acids, glutathione metabolism, and purine and pyrimidine metabolism showing severe perturbations.

• Lipid perturbations were also investigated with untargeted lipidomics, although changes at this early time point were not as pronounced as seen with metabolomics. However, GCR+HU led to decreases in overall sphingomyelins.

Aim 2 focused on determining the extent of perturbations, and whether those are seen to exert additive or synergistic effects.

• Fold changes and combination indices were calculated for metabolites, specifically involved in energy metabolism with mitochondrial involvement, such as carnitine, acetylcarnitine, and tricarboxylic acid (TCA) cycle intermediates.

• Furthermore, cofactors and vitamins were quantified in muscles with semi-quantitative targeted liquid chromatography methods.

• As seen with spaceflight experiments, select cofactors such as previously reported pantothenic acid, show consistently altered levels in ground-based studies, as in spaceflight.