We analyzed the muscle biopsies of two astronauts who spent six months on the International Space Station (ISS) using mass spectrometry-based proteomics, with the aim of assessing how spaceflight modifies the structure and function of human skeletal muscle. We quantified on average 3800 proteins per sample, and a total of over 7000 proteins, spanning seven orders of magnitude in intensity. The two astronauts trained onboard using a treadmill and an Advanced Resistive Exercise Device (aRED) to simulate the use of free weights in the absence of gravity. The astronaut A trained more intensively and could preserve 80% of his muscle fiber cross-sectional area, compared to only 50% of astronaut B. Comparing the biopsies taken pre-mission to those from the day of landing we revealed in both astronauts a dramatic decrease in the expression of mitochondrial proteins located in all main compartments of the organelle, particularly the inner mitochondrial membrane and the matrix. The correlation of the mitochondrial protein loss to the relative amount of physical exercise was less straightforward than for the preservation of muscle mass. Our results are in line with the emerging consensus that mitochondria are a hub of the impact of spaceflight in humans as well as in various model organisms that were stationed on the ISS. Our data show that the relationship between exercise, muscle growth and mitochondrial biogenesis works differently in the space environment. My talk will discuss reactive oxygen species as a possible direct cause of mitochondrial damage and propose a role of physical exercise in the induction of an antioxidant response which can partially safeguard skeletal muscle structure and function.