Introduction <p>Saliva biomarker research requires understanding of how saliva collection and processing techniques affect results and reproducibility. Direct comparison of the effect of processing and storage conditions on downstream assay outcomes is important to understanding the ways that delay in freezing, methods of freezing, filtration protocols, and other factors can affect results and thus, interpretations about stress response and adaptation. This type of study compliments the work that is required to understand how saliva compares to tissue and blood responses and what the implications of changes in saliva biomarkers mean to our understanding of biomarkers of stress during exercise and environmental stress exposures.</p> Objective <p>In this pilot study, we evaluated the effects of different processing methods on metabolomics results and discuss the implications of these findings for future experimental design.</p> Methods <p>We assessed the effects of centrifugation, time-of-day collection, filtration, and mucinase treatments and determined that there are quantifiable differences in metabolomics results with different treatments.</p> Results <p>Adding processing steps did not increase the number of metabolites detected or the sensitivity and specificity of results.</p> Conclusion <p>Comprehensive descriptions of methods in this area will support better interpretation and reproducibility in this field.</p>