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Long-duration spaceflight impacts human physiology, including well documented immune system dysregulation. Diet, the microbiome, and immune system function are interlinked, but diet is the only one of these factors that we have the ability to easily, and significantly, alter on Earth or during flight. As we better understand dietary impacts on physiology, we may then improve the spaceflight diet to improve crew health and potentially reduce spaceflight-associated physiological decrements. Increasing the consumption of fruits and vegetables and bioactive compounds (e.g., omega-3 fatty acids, lycopene, flavonoids) and therefore enhancing overall nutritional intake from the nominal shelf-stable, fully-processed, space food system is expected to serve as a countermeasure to detrimental impacts to human physiology, including dysregulation in immunological profiles, the taxonomic profile of the gut microbiota, and nutritional status during spaceflight. In this study, first we sought to determine the effect of the nominal shelf-stable spaceflight diet compared to an "enhanced" shelf-stable spaceflight diet on human biochemistry, immunology, and the microbiome in a ground-based, simulated space mission. The ground analog portion of this study was conducted in the NASA Human Exploration Research Analog (HERA) Campaign 4 missions, which consisted of four 45-day missions with closed chamber confinement and realistic mission simulation to study effects on crew health and performance. As reported previously, analyses indicate beneficial associations between diet and markers of nutritional status, stress, the microbiome, and cognitive performance. Intake and beneficial associations varied by subject. This data will be used as a ground-based control for spaceflight, where the spaceflight environment (e.g., radiation, microgravity) will have additional impacts and the potential to evaluate effects of the diet will be greater. The second phase of this study is to occur on the International Space Station, where it is currently being implemented. The test plan is similar to that used in the HERA missions. The enhanced diet is intended to provide 25% of the crews’ diet with foods rich in omega-3 fatty acids, lycopene, and flavonoids, along with more fruits and vegetables in general (the other 75% of the diet will be obtained from standard and crew preference items available on the ISS). Biological samples (blood, urine, stool, and saliva) are being collected from participants at selected time points before, during, and after the mission. Data collection also includes dietary intake recording and body mass measurement. Currently, 6 of 9 planned astronauts have completed data collection. Analysis of immune markers, latent herpes virus reactivation, the taxonomic and metatranscriptomic profile of the gut microbiome, and nutritional status biomarkers and biochemical metabolites will occur in batch to minimize sample handling variations. Mixed models statistical analyses will be used, incorporating random effects to account for repeated measures within individuals to assess the impact of diet on physiological outcomes. We expect this study to provide evidence of beneficial impact of this enhanced diet on crew health and adaptation to spaceflight. These data will aid in evidence-based mass-risk trades for food system design and development of targeted dietary interventions for future exploration-class space missions.