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Description: Although some studies reveal that saliva handling and storage practices may influence salivary testosterone concentrations measured with immunoassays, the effect of these methodological factors on the validity of testosterone immunoassays remains unknown. The validity of immunoassays can be assessed by comparing hormone concentrations measured with immunoassays to a standard reference method: liquid chromatography tandem mass spectrometry (MS). We previously reported the correspondence between salivary testosterone measured with enzyme immunoassays (EIAs) and with MS when methodological factors related to saliva handling and storage were tightly controlled across measurement methods (Welker et al., 2016). In the present study, we expanded the original dataset and compared the correspondence between Salimetrics EIAs and MS when variance in these methodological factors across measurement methods was tightly controlled (low methodological variance) to when variance in these factors was less controlled (high methodological variance). If increased variance in these methodological factors impacts the validity of testosterone measurement, then the EIA-MS correspondence should be stronger under conditions of lower rather than higher methodological variance. Our results contradicted this hypothesis: the Salimetrics EIA-MS correspondence was stronger when there was higher, compared to when there was lower, variance in these methodological factors. A potential explanation for this instability in EIA-MS correspondence could be variance in antibody performance in EIA kits. Although the average correlation across low and high methodological comparisons provides an updated estimate of the Salimetrics EIA-MS correspondence, the instability in this correspondence may pose direct challenges to the reproducibility of psychoneuroendocrinology research.