Although many aspects of microbiome studies have been standardized to improve experimental replicability, none account for how the daily diurnal fluctuations in the gut lumen cause dynamic changes in 16S amplicon sequencing. Here we show that sample collection time affects the conclusions drawn from microbiome studies and are larger than the effect size of a daily experimental intervention or dietary changes. The timing of divergence of the microbiome composition between experimental and

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... groups are unique to each experiment. Sample collection times as short as only four hours apart lead to vastly different conclusions. Lack of consistency in the time of sample collection may explain poor cross-study replicability in microbiome research. Without looking at other data, the impact on other fields is unknown but potentially significant.