Aim: We investigated the effect of pre-analytical sample handling variations on coronavirus disease 2019-relevant circulating cytokine levels IFN-gamma, IL-10, IL-12p70, IL-17A, IL-6 and TNF-alpha. Materials & methods: We collected blood in different collection tubes (ethylenediaminetetraacetic acid, sodium citrate, lithium heparin, serum), and subjected ethylenediaminetetraacetic acid plasma to among others increasing delays in centrifugation or -80 degrees C storage. Six subjects were included in each experimental condition. Cytokine levels were measured in these samples using the Simoa Cytokine 6-plex kit. Results: Different tube types resulted in different blood cytokine levels. IL-17A and IL-6 levels declined with 3 h centrifugation delay. IFN-gamma levels declined with 24 h postcentrifugation storage delay. IL-17A levels declined with 2-week storage delay. Conclusion: It is recommended to centrifuge tubes quickly following collection, for accurate cytokine measurement.