Updated project metadata. Spaceflight causes loss of muscle mass and strength, metabolic remodeling and insulin resistance, contributing to severe challenges for astronaut health. We used highly sensitive proteomics to detail single fiber type-specific molecular remodeling caused by muscle unloading in subjects undergoing prolonged bed rest. We then measured the muscle proteome of astronauts before and after a mission on the ISS. Our combined datasets show downregulation of complexes involved in fiber-matrix interaction, force transmission and insulin receptor stabilization on the sarcolemma. Unloading upregulated anti-oxidant responses in slow but not in fast fibers, markers of neuromuscular damage and the hypusination pathway that uniquely modifies translation initiation factor 5A. These muscle intrinsic changes in adhesion, protein turnover and redox balance may contribute to the whole-body detrimental effects caused by prolonged unloading. We conclude that single muscle fiber analysis by proteomics can support the development of advanced countermeasures targeting the mechano-metabolic molecular axis.