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Objectives: To evaluate the reliability of data from the assay of bio‐archived specimens, a 50‐freeze–thaw‐cycle (FTC) degradation study of fresh sera was conducted to test the stability of 16 immunoregulators. Methods: Twenty de‐identified serum specimens were obtained from volunteers at United Health Services‐Wilson Memorial Hospital. Specimens were stored at −20°C and underwent daily 1 h thawing and subsequent freezing for each FTC over 50 consecutive days. Immunoregulator concentrations were assessed via enzyme‐linked immunosorbent assay (ELISA) in participant samples at 2 FTC (baseline), 25 FTC, and 50 FTC. Specific immunoregulators observed in the study were C‐reactive protein (CRP), interleukin (IL)‐1α, 4, 6, 8, 10, monocyte chemoattractant protein‐1 (MCP‐1, CCL2), monocyte chemoattractant protein‐2 (MCP‐2, CCL8), eotaxin‐1, thymus‐and‐activation‐regulated chemokine (TARC, CCL17), regulated on activation normal T‐cell expressed and secreted (RANTES, CCL5), growth‐regulated oncogene‐alpha (GRO‐α, CXCL1), small inducible cytokine A1 (I‐309, CCL1), interferon‐gamma (IFN‐γ), interferon‐gamma inducible protein‐10 (IP‐10, CXCL10), and tumor necrosis factor‐alpha (TNF‐α). Results: Quantitative stability of serum immunoregulators: Serum CRP, IL‐8, IL‐10, IFN‐γ, IP‐10, and eotaxin‐1 levels appear to be statistically equivalent from baseline to 50 FTC (p ≤.05). Retention of patterns in serum immunoregulators: patterns across FTC were retained for TARC (age) and CRP, IFN‐γ, and MCP‐2 (sex). Conclusions: While the effect of multiple FTC on serum immunoregulator levels may not replicate prolonged freezer storage, the results of this study provide valuable information on the robustness of immunoregulators for research using bio‐archived sera. [ABSTRACT FROM AUTHOR]