AbstractMinimizing variability in collection and processing of human blood samples for research remains a challenge. Delaying plasma or serum isolation after phlebotomy (processing delay) can cause perturbations of numerous analytes. Thus, a comprehensive understanding of how processing delay affects major endpoints used in human immunology research is necessary. Therefore, we studied how processing delay affects commonly measured cytokines and immune cell populations. We hypothesized that short-term time delays inherent to human research in serum and plasma processing impact commonly studied immunological analytes. Blood from healthy donors was subjected to processing delays commonly encountered in sample collection, and then assayed by 62-plex Luminex panel, 40-parameter mass cytometry panel, and 540,000 transcript expression microarray. Variance for immunological analytes was estimated using each individual’s baseline as a control. In general, short-term processing delay led to small changes in plasma and serum cytokines (range − 10.8 to 43.5%), markers and frequencies of peripheral blood mononuclear cell phenotypes (range 0.19 to 3.54 fold), and whole blood gene expression (stable for > 20 K genes)—with several exceptions described herein. Importantly, we built an open-access web application allowing investigators to estimate the degree of variance expected from processing delay for measurements of interest based on the data reported here.