<p>Microgravity is a condition that may affect gastrointestinal function and metabolism during spaceflight. Despite attempts to keep normal dietary habits at the ISS, the food selection and meal timing may also be perturbed during the confinement. To understand the impact of spaceflight on human metabolism, we characterized the plasma metabolome of astronauts (<i>n</i> = 52) before, during, and after missions on board the International Space Station (ISS) using liquid chromatography coupled with mass spectrometry. Here we show that spaceflight affected around 40 circulating compounds. In this longitudinal assessment, the metabolic changes were already observed shortly after launch and subsided within a few days after landing. The flight-induced changes in metabolites reflected increased protein fermentation by the gut microbiota, possibly reflecting prolonged intestinal transit time caused by microgravity. Minor diet-related changes related to intakes of caffeine, fish, and some fats were also observed but affected less than one third of all metabolites responding to spaceflight. We did not observe major sex-specific differences in metabolism. Increasing the consumption of slow-fermented carbohydrates at ISS to reduce protein fermentation might improve gastrointestinal health in astronauts in future human long-term spaceflights.</p>