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The microbiome has immense power to shape health outcomes. The bacteria that take up residence in the human body, including the gut, have been shown to impact health and disease, including neurodegenerative disorders. Medical research is invested in finding ways to reconstruct healthy microbiomes to help cure some of those ills. 

But like many fields in science, microbiome research is suffering from a reproducibility crisis. Eating, sleeping, and exercising can all affect the types and amounts of bacteria in the body, which can in turn make it difficult to replicate experimental outcomes. And in a new study, researchers highlight yet another confounding factor contributing to the problem: time.


“The microbiome is constantly changing,” said Amir Zarrinpar, a gastroenterologist and gut microbiome researcher at the University of California, San Diego. But microbiome research, in which the bacteria in a sample are identified and quantified through sequencing, can only capture snapshots. In a study published on Monday in Nature Metabolism, Zarrinpar and his colleagues argue that the circadian rhythm and its impact on the microbiome affects research reproducibility, and should be reported in experimental studies.  

“When we look through papers in the literature, people don’t report what time of day the experiment was done, which means they are not controlling for it,” said Zarrinpar. He and his colleagues analyzed bacterial sequencing data published in 550 articles since 2019 to find that only 0.32% of microbiome papers reported time of day. Then, they dug into the few datasets that did report time to determine just how much of an impact it could have on microbiome composition.

The researchers analyzed bacterial sequencing data from prior publications that reported circadian rhythm based on when the lights in the mice housing facilities were switched on or off and the multiple times at which the data were collected. The researchers showed that for samples taken at intervals as few as four hours apart, the microbiome composition changes drastically.


Zarrinpar’s team theorized that a mouse’s circadian rhythm — primarily measured by behavioral cues, such as when mice are eating, active, and sleeping — changes the composition and amount of bacteria present. 

There are other variables that can have a major impact on microbiome composition. Food, especially, can impact the microbial communities that flourish in the gut, said Molly Bray, a circadian rhythm researcher at the University of Texas at Austin who was not involved in the study. She agreed that time was a critical factor to consider.

But the researchers were surprised that circadian rhythms appeared to have even more of an impact on the microbiome than diet. “This is not some small effect — this is just as big as diet,” Zarrinpar said. 

There could be some interplay between diet and circadian rhythms, though, scientists say — it’s possible that eating at different times of the day could favor growth of different bacteria.

The microbiome doesn’t just affect the gut, but other organs as well. Germ-free mice, mice without an intact microbiome, are more susceptible to liver disease. 

Additionally, “every organ has a circadian clock and a way of keeping time,” said Zarrinpar. His team didn’t report on organ-specific activity, but he noted that such variability can also affect experimental results. 

No matter the mechanism, Zarrinpar argues that especially for microbiome studies, scientists need to take into account time of day. He and his colleagues presented a set of guidelines to help researchers conduct such studies.

That’s easier said than done, of course. “The best way to document the effects of circadian variability, especially in terms of the gut microbiome, is to collect multiple samples over the course of the day and to analyze the data as a whole, not just by time point,” said Bray in an email to STAT. That’s possible for animals — just pick up their poop multiple times a day — but much harder and more expensive when trying to conduct gut microbiome research in humans. Consider trying to study skin, vaginal, and oral microbiomes and the challenges multiply.

And accounting for changes in the circadian rhythm when collecting data samples from humans will be challenging.  “It’s doable to collect feces over the course of the day in animals,” Bray noted, “but not so easy (or even possible) in humans.” 

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