Stervbo U, Bajda S, Wehler P, et al. - Cytokines serve in diagnosis and monitoring of immune-related disorders, for which, there is the requirement for strictly controlled handling of the samples including storage and freeze/thawing procedures. In basic research and clinical trials, long-term storage of human serum samples is done before processing. There is a limited storage space in scientific laboratories as well as there existed practical limitations to the number of analytes to be processed at the same time. New findings and technological progress might provoke analysis of hitherto unconsidered or undetectable molecules. Therefore, a likely scenario is repeated freeze/thawing of serum samples, raising the question of the stability of the measured analytes under such conditions. Researchers here addressed this question via subjecting serum samples with spiked-in T-helper cell linked cytokines to several cycles of freeze/thawing under various conditions, including storage at −20 °C or −80 °C and thawing at 4 °C, 22 °C, and 37 °C, respectively. Storage at room temperature for 4 h before freezing led to a decrease in the concentration of TNF-α, IL-4, IL-17F, and IL-22. Generally, there was a reduction in cytokine concentrations when stored at −20 °C. This opposes storage at −80 °C, which produced stable analyte concentrations; unaffected by repeated freeze/thaw cycles. Findings thereby highlight the requirement for sentinel samples with known cytokine concentrations as internal control for the freeze/thaw process.