Given the limited knowledge of the biological impact of spaceflight, a multi-omics, systems biology approach was used to investigate NASA’s GeneLab data and astronaut biomedical profiles. These data consist of hundreds of samples flown in space, human metrics from 59 astronauts, and confirmatory data from NASA’s Twin Study, analyzed together for consistent transcriptomic, proteomic, metabolomic, and epigenetic response to spaceflight. Pathway analysis showed significant enrichment of mitochondrial activity and innate immunity. Muscle and liver tissues showed that chronic inflammation, may be a response to mitochondrial dysfunction. Additional pathways altered in spaceflight included cell cycle, circadian rhythm, and olfactory activity pathways, all of which are known to have interactions with mitochondrial activity. Evidence of altered mitochondrial function was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, all of which indicate mitochondrial stress as a consistent phenotype of spaceflight.