This observational follow-up study extends a previously completed 15-day intensive longitudinal study (WeF) of the gut microbiome in healthy adults. The objective of WeF2 is to characterize the long-term stability and dynamics of the gut and oral microbiota in the same cohort across multiple timescales, and to determine whether the day-to-day variability patterns observed at baseline persist over months and years. Participants who completed the WeF baseline protocol will be invited to attend periodic follow-up visits for low-frequency biospecimen collection and to repeat the original 15-day high-frequency sampling protocol at planned intervals, beginning approximately six months after the WeF baseline and continuing at extended intervals thereafter. By combining sparse routine sampling with recurring high-density sampling windows, the study aims to distinguish the stable core microbiome from transient and seasonal fluctuations, to track gradual community shifts under free-living conditions, and to further explore relationships among microbiome dynamics, dietary intake, and host glycemic regulation over an extended observation period.

WeF2 is a non-interventional, observational follow-up study conducted in the cohort of healthy adult volunteers who completed the WeF baseline protocol. The study is designed to extend the temporal resolution of the original investigation from a single 15-day window to a multi-year horizon that interleaves low- and high-frequency sampling phases, allowing direct comparison of within-subject microbiome dynamics across short, medium, and long timescales. The overall follow-up duration is open-ended in design, with the intention that participants may continue to contribute to the cohort for as long as they remain willing and eligible.

The study is organized around two complementary sampling modes that recur throughout the follow-up period.

The routine follow-up mode consists of brief periodic visits, planned at approximately monthly intervals during the intervals between intensive sampling windows. At each routine visit, a single stool sample and a saliva sample are collected for microbial sequencing and functional evaluation, together with basic health and lifestyle information covering items such as recent dietary patterns, medication and supplement use, antibiotic exposure, illness, travel, and notable lifestyle changes since the previous visit. The exact content and cadence of these routine visits may be adjusted over the course of the study to reflect emerging scientific priorities, participant burden considerations, and operational feasibility, while preserving the core objective of capturing gradual community shifts and flagging transient perturbations occurring between intensive windows.

The intensive sampling mode replicates the original 15-day WeF protocol in full and is scheduled to recur at planned long-interval timepoints. The first intensive follow-up window is conducted approximately six months after completion of the WeF baseline; a second intensive window is planned approximately six months after the first (i.e., approximately one year after the WeF baseline); and additional intensive windows may be conducted at extended intervals thereafter as the cohort is maintained. Each intensive window involves daily collection of stool and saliva samples; collection of representative samples from meals consumed during the period for DNA sequencing of food sources, providing an objective record of dietary intake; and continuous blood glucose monitoring via a wearable CGM device throughout the 15 days. By holding the intensive protocol methodologically consistent with the WeF baseline, each intensive window produces a directly comparable dataset at the level of individual taxa, community structure, dietary signatures, and glycemic responses.

Routine follow-up visits continue in the periods between intensive windows, including after each intensive window has concluded, so that the cohort is observed under a continuous low-frequency framework punctuated by recurring high-frequency snapshots.

Integrating these sampling modes produces a longitudinal dataset that supports several complementary analyses: characterization of the temporal stability of the core microbiota across months and years; assessment of whether the magnitude and structure of day-to-day variability observed at baseline are reproducible at later timepoints; differentiation of intra-individual fluctuations from inter-individual differences across an expanded observation period; identification of seasonal or longer-cycle patterns in microbiome composition; and exploration of how dietary inputs and glycemic patterns relate to microbial dynamics on daily, monthly, and annual scales.

To ensure data integrity and participant safety, the study is overseen by a Data and Safety Monitoring Board and adheres to ethical standards approved by the Westlake University Ethics Committee, consistent with the framework established for the WeF baseline study.

Inclusion Criteria:

• Participants reside in Hangzhou

Exclusion Criteria:

• Refusing or unable to give informed consent
• Participants with chronic gastrointestinal diseases and take daily relevant medications
• Participants with metabolic diseases including diabetes, hypertension and cardiovascular diseases (CVD)
• Participants with craniocerebral trauma, cancer, liver disease, kidney disease, or other critical illness, or history of operation or medication
• Participants with bulimia nervosa, post-traumatic stress disorder (PTSD), chronic anxiety and depression or other critical neuronal disorder or history of relevant medication
• Being or to be pregnant or lactating.
• Participants with history of alcohol or drug addiction, or smoke above 15 cigarettes per day
• Concurrently participating other clinical trials.

Healthy young adults residing in Hangzhou