Abstract Background: Serologic testing for antibodies against epitopes from pathogens is a valuable tool for investigating the relationship between infection and disease. This study comprehensively evaluates the impact of preanalytic variation on antibody seropositivities to a selected set of antigens arising from delays in processing of blood samples, preprocessing storage temperature, and vacutainer type. Methods: We assessed peripheral blood collected from 29 volunteers in four different Vacutainer types [ethylenediaminoetetraacetic acid (EDTA), acid-citrate-dextrose (ACD), lithium heparin (LH), serum separator tubes (SST)], and stored at 4°C or room temperature for 0, 1, 2, 3, 4, 5, and 6 days before processing. Multiplex serology was used to determine antibody reactivity against 35 antigens derived from human papillomaviruses, human polyomaviruses, Epstein–Barr virus, and Helicobacter pylori. Cohen's κ statistic was used to measure agreement on seropositivity status between samples exposed to standard and nonstandard clinical practice conditions. Results: For samples processed without delay, κ was not associated with storage-temperature (P value range 0.23 to 0.95) or vacutainer type (P value range, 0.35–0.89). Kappa did not significantly decline with increasing delays in processing for any vacutainer-type storage temperature combination (P slope range, 0.06–1.00). Conclusions: Antibodies to epitopes from various pathogenic infectious agents can be measured reliably from samples stored in SST, EDTA, ACD, or LH vacutainers at either room temperature or 4°C for up to 6 days before processing. Impact: Serologic testing is robust to several preanalytic options. These findings are particularly important for epidemiologic studies recruiting participants from remote settings where sample exposure to preanalytic conditions can vary considerably. Cancer Epidemiol Biomarkers Prev; 26(8); 1337–44. ©2017 AACR.