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 k 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, k 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.