Background: Serum measurements of cytokines, mediators of various B and T cell activities, are important markers of inflammation and immune dysregulation. We assessed the reproducibility of serum cytokine measurements over a five-year period among participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Methods: Levels of 13 cytokines [interleukin (IL) 1 beta, IL-2, 1L-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, interferon-gamma (IFN gamma), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF alpha)] in stored sera from three collections (study baseline,+1 year, and + 5 years) among 28 randomly selected PLCO participants were measured using a high-sensitivity Luminex xMap-based multiplex panel. Within-and between-subject components of variance were estimated from random effects models and were used to calculate the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for analytes with <30% of samples below the limit of detection (LOD). Spearman correlation coefficients between measurements of the same analyte over time and between analytes were also calculated. Results: Among the six cytokines with <30% of samples below the LOD, we observed excellent reproducibility for IL-6, IL-7, IL-13, and TNF alpha (ICC >= 0.73), and fair to good reproducibility for IL-8 (ICC = 0.55) and IL-10 (ICC = 0.60). Spearman correlation coefficients comparing paired measurements of each cytokine at baseline and at +5 years were high (p >= 0.74) with the exception of IL-10 (p = 0.44). Conclusions: These results suggest that measurements of most of the cytokines evaluated in this study were highly reproducible over five-year periods. Published by Elsevier Ltd.