Fig. 1. Study design and human spaceflight metrics.

The flight subject (TW, blue) and his identical twin (HR, green) were each studied over 25 months using a comprehensive set of health and biological metrics. (A) Earth-based and spaceflight collections include blood, sorted cells (CD4, CD8, CD19, and LD), stool, buccal, saliva, urine, and AR blood. TCR, T cell receptor sequencing; T- and U-Metabolomics, targeted and untargeted metabolomics; OSI, oxidative stress and inflammation; T- and U-Proteomics, targeted and untargeted proteomics; CVU, cardiac and vascular ultrasound; VSF, vascular structure and function. (B) Gene expression changes in TW inflight and postflight compared with the preflight period. All time points of HR were used to account for normal levels of variance and noise in gene expression. Genes that were significantly altered inflight after controlling for subject baselines and AR effect are reported. Gene expression changes were reported for any gene with q < 0.05 in a multivariate model that utilized expression values for polyadenylated [poly (A)⁺] and ribosomal RNA depleted transcripts. (C) Metabolites present at significantly different levels in HR and TW or between pre-, in-, and postflight periods. Heatmap represents median-normalized log₂ intensity for each analyte, scaled across all samples. Red color indicates relative enrichment, whereas blue indicates relative depletion. (D) GO analysis of genes ranked on the basis of epigenetic discordance at their promoters. Comparisons of preflight samples to inflight (early and late, and combined early and late) and postflight samples are shown for both CD4 and CD8 cells. Heatmaps represent transformed enrichment values [square root (sqrt) of enrichment] for GO categories with a raw enrichment value >5. Reg., regulation; neg. reg., negative regulation. (E) C-means clustering of multiomics data reveals longitudinal patterns associated with spaceflight. Analyte abundance (scaled) plotted over time for the identified clusters from the integrated metabolome, proteome, cytokine, cognition, and microbiome datasets is shown. Median abundance (bold) per cluster and 5th and 95th percentiles of abundance (shaded) are indicated. The gray shaded region indicates the inflight period. n, number of analytes in cluster. (F) Individual plots of the different analyte types that compose the spaceflight-dependent cluster, cluster 3, from (E). Telomere levels are plotted adjacent to the cluster members for reference. Median abundance (bold) per cluster and 5th and 95th percentiles of abundance (shaded) are indicated. Thin lines show annotated examples of analytes from cluster 3. For Cognition, accuracy [MP(Acc)] and speed [MP(Spd)] on the motor praxis task as well as standardized speed across cognitive domains (speed) are shown. n, number of analytes in cluster; CAG, coabundance gene groups.