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Background:In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at −80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for saliva) among 51 healthy volunteers.Methods:Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the 20 most abundant genera, three alpha-diversity metrics, and the first principal coordinates of three beta-diversity matrices.Results:The subject variability was much higher than the variability introduced by the sample collection type, and storage time. For fecal samples, microbial stability over 2 years was high across collection methods (range, ICCs = 0.70–0.99), except for the samples collected with no additive (range, ICCs = 0.23–0.83). For oral samples, most microbiome diversity measures were stable over time with ICCs above 0.74; however, ICCs for the samples collected with Scope mouthwash were lower for two alpha-diversity measures, Faith's phylogenetic diversity (0.23) and the observed number of operational taxonomic units (0.23).Conclusions:Fecal and oral samples in most used collection methods are stable for microbiome analyses after 2 years at −80°C, except for fecal samples with no additive.Impact:This study provides evidence that samples stored for an extended period from prospective studies are useful for microbiome analyses.