Effects of passive fluid restriction and rehydration protocols on circulating immune cell transcriptomics over multiple consecutive days

Dehydration diversely affects on health and performance. The effects of whole body dehydration on intracellular volume and plasma osmolality are limited as those compartments are tightly protected, but small changes in plasma osmolality even up to ~20mOsm×kg-1 (milliosmoles per kilogram) may affect circulating immune cells (peripheral blood mononuclear cells, PBMCs) in ways that alter global gene expression landscapes that may impact resiliency or susceptibility to infection acutely and with chronic stress. Thus, our study aim was to 1) quantify hypertonicity experienced by circulating peripheral blood mononuclear cells and neutrophils during passive dehydration and rehydration protocols administered to healthy participants over multiple, consecutive days, and in that context, 2) quantify genome-wide transcriptomic changes in cell lysates using RNAseq. 18 healthy, male study volunteers (23±3.28 years, 80.09±9.6 kg, 175.78±5.68 cm) participated in our study with 3-5 (Days -5 to 0) days of baseline testing and establishment of euhydrated state measures as confirmed by urine, plasma, and acute body mass changes, per individual. Day 1 designated the first day that participants were euhydrated and on Day 2 asked to commence a 24-hour passive dehydration protocol during which participants restrict fluid consumption to induce levels of whole-body dehydration that correspond to ~ 2-4% relative body mass loss in water. A repeated measures analysis of variance was used to analyze the differences across timepoints for plasma osmolality nad other hydration measures. RNAseq was conducted (Illumina NextSeq 500) to achieve 40-45 million paired end reads (2x75bp (base pair)) per sample at 5 time points during which participants were euhydrated (EUH), partially euhydrated from rehydration with water (PARTEUH), or hypohydrated (PARTDEHY, partial dehydration or DEHY, full dehydration achieved with protocol): Days 2AM (EUH), 2PM (PARTDEHY), 3AM (DEHY), 3RH (PARTEUH), 4AM (EUH). Standard RNAseq analysis and functional annotation pipelines were used to determine differentially expressed genes and associated functions between time points within subjects. ANOVA revealed significant group by time interaction with respect to plasma osmolality (F=4.960, p< 0.00001, observed power=.996). Differentially expressed gene numbers were 350, 961, 334, and 109 for comparisons between Day2AM and 3AM, 2AM and 2PM, 2AM and 3RH, and 2AM and 4AM, respectively. In the most pronounced difference (2AM vs. 3AM), upregulated biological process GO Terms included coagulation, humoral immune response, granulocyte activation, neutrophil-mediated immunity, and regulation of body fluid levels. Downregulated biological process GO Terms included response to interferon-gamma, interferon-gamma-signaling pathway, innate immune response, and lymphocyte mediated immunity. Ongoing work is determining functional and pathway annotation of comparisons between hydration states and examining specific genes to determine their candidacy as key regulators of immune response during hydration stress or novel biomarkers related to dehydration. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.