Ethical statements
The study protocol was approved by the ethics committees of all participating institutions, including the National Center for Global Health and Medicine (NCGM-S-004586), Tokyo Medical University Hospital (E2023-0026), University of Tokyo (No. 68-Multicenter), Hitachi Health Care Center (2021-33), Institute of Health Sciences, Ezaki Glico (2023-23-240913) and the RIKEN Center for Integrative Medical. Science (2023-21). The study was conducted in accordance with the Declaration of Helsinki and relevant ethical guidelines. Written informed consent was obtained from all participants before enrollment. No financial compensation was paid to participants for their participation in this study.
Japanese 4D microbiome project
The Japanese 4D Microbiome Project is a multi-center study involving hospitals, medical screening centers and research institutes across Japan. Collection of data and biological samples began in January 2015 and continues. Participants were recruited from people undergoing routine health examinations or clinical assessments. Clinical and lifestyle metadata were collected from healthy and diseased individuals using standardized questionnaires, structured interviews, and electronic medical records.26. Stool samples were collected along with metadata from all participants, and blood samples were obtained from individuals who provided additional consent.
Eligibility criteria included: (1) self-reported Japanese ethnicity and residence in Japan; (2) age ≥16 years, with no upper age limit; and (3) provision of written informed consent. Exclusion criteria included: (1) acute gastrointestinal hemorrhage; (2) conditions that prevented completion of questionnaires or giving informed consent; and (3) significant limitations in activities of daily living.
In the overall 4D cohort, 6,803 individuals with fecal samples and corresponding metadata were included in the cross-sectional analysis (Extended Data Fig. 1). Additionally, a longitudinal analysis was conducted on a subset of 402 individuals to assess intra-individual changes in the gut microbiome. These participants provided a total of 804 stool samples collected at two time points, with a median interval of 2.0 years between collections. Lifestyle information was assessed at both time points using standardized questionnaires, as described below.
Collection of metadata and variables of interest
Participants completed a self-administered questionnaire covering lifestyle habits, medical history, comorbidities and medication use.26. Research assistants then reviewed the responses with each participant to correct inaccuracies and minimize missing data. Clinical information on physician-confirmed diagnoses and prescribed medications was also abstracted from electronic medical records by trained staff.
In this study, modifiable lifestyle factors, including diet, alcohol consumption, smoking, physical activity and sedentary behavior, were considered the main variables of interest, alongside health status and disease. Other variables, including age, sex, BMI, and medication use, were treated as covariates. Diseases with fewer than 100 cases were excluded from the primary analysis. Medication use was not included as a primary variable, but was considered in the definition of general health status and as a covariate in multivariate analyses. Sex was obtained primarily from medical records of participating hospitals and health facilities and supplemented by self-reported questionnaire data. The sex recorded in the medical record reflected the medically documented sex of the participant. Gender identity was not collected separately.
Assessment of lifestyle factors
Lifestyle factors, including dietary habits, alcohol consumption, smoking, physical activity, and sedentary behavior, were assessed using a self-administered questionnaire. All lifestyle variables were assessed at the time of sample collection. Eating habits and alcohol consumption over the past month were assessed using a self-reported meal frequency questionnaire, with items rated on a 7-point Likert scale (1, never or rarely; 2, one to three times a month; 3, one to three times a week; 4, four to six times a week; 5, once a day; 6, twice a day; and 7, three or more times a day).26. Dietary variables were then grouped into predefined categories (e.g., plant-based diets, animal-based diets, fermented foods, and refined carbohydrates). Alcohol consumption was then quantified by recording the quantity consumed per occasion and weekly frequency, from which weekly alcohol consumption (grams per week) was calculated. Smoking status was defined based on current smoking behavior.
Physical activity was assessed using the International Physical Activity Questionnaire-Short Form (IPAQ-SF), an instrument validated for adult populations.61. Activities were categorized into three intensity levels: low (e.g., leisurely walking), moderate (e.g., brisk walking, recreational cycling), and high (e.g., running, competitive sports). Prolonged sedentary behavior (sitting time) was defined as ≥9.5 hours per day, a threshold associated with a marked increase in risk of all-cause mortality in a nonlinear dose-response relationship.4.
Definition of general health and diagnosis of chronic diseases
Individuals classified as having good general health (n= 2,119) were defined as those without comorbidities, without a history of chronic diseases or surgery, without taking medications, or without infection with hepatitis B virus, hepatitis C virus, or HIV. To ensure a strict definition of general health, medication use was assessed across 895 medication categories, and individuals reporting medication use were not classified as having general health.
Self-reported diagnoses of chronic conditions have lower sensitivity than physician-confirmed diagnoses, which are considered the gold standard.26. This discrepancy is because some people may not report the conditions they actually have and is particularly pronounced for heart failure, connective tissue disease, and chronic obstructive pulmonary disease.26. Therefore, to ensure diagnostic accuracy, physician-confirmed diagnoses were mainly used in this study. Diagnoses of chronic diseases were based on typical clinical symptoms and supported by the results of endoscopic examinations, histopathological analyses, imaging studies (computed tomography, magnetic resonance imaging, or ultrasound), and blood tests. All diagnostic data were obtained from electronic medical records at each participating institution. For example, IBD was diagnosed based on endoscopic findings and biopsies, with infectious enterocolitis ruled out through negative culture tests and clinical courses. In contrast, IBS was diagnosed using self-reported questionnaires based on the Rome IV criteria.
Medication Utilization Assessment
Medication use was assessed by trained research staff through review of self-administered questionnaires, electronic medical records, and medication history notebooks (“Okusuri Techo”), which are maintained by pharmacists to document prescription details.26. Medication use was defined as use of oral or self-administered injectable medications in the past month, and this definition was applied consistently across all analyses. All drugs were classified according to the World Health Organization Anatomical, Therapeutic and Chemical (ATC) classification system.
Confounding factors
To control for potential confounds, all association analyzes between host-related variables (including lifestyle factors and clinical outcomes), gut microbiome characteristics, and microbiome-based scores were adjusted for a predefined set of covariates. These covariates were selected based on known factors associated with both host and gut microbiome variables and included eight covariates: age, sex, BMI, and five medication use variables. Medication use was defined as the use of medications known to significantly influence the gut microbiome.26including proton pump inhibitors, antibiotics, statins, antidiabetic drugs (metformin), and antihypertensive agents (angiotensin II receptor blockers).
To ensure model stability, the number of covariates included in the multivariate models was limited according to the rule of 10 events per variable.62. Based on the minimum number of outcome events (≥100 cases), the number of covariates was limited to a maximum of 10 and eight covariates were included. Although moderate correlations were observed for a small number of exposure pairs (e.g. vegetables and mushrooms, vinegar and mushrooms), most exposure pairs had low correlations (Spearman’s rho < 0.4; Supplementary Table 2). Therefore, to minimize multicollinearity and improve the interpretability of exposure-microbiome associations, lifestyle variables were analyzed individually.
Analyzes involving host metadata, including microbial diversity analyses, MaAsLin2 models, and mediation analyses, were adjusted for eight covariates. In contrast, correlation analyzes between bacterial species and functional traits (KO, SCFA, and cytokines) were performed without covariate adjustment, as these analyzes were exploratory and aimed to characterize interomic relationships.
Fecal sample collection
Fecal samples were collected at home by participants and submitted to the study facilities on the same day, whenever possible. If same-day submission was not possible, samples were stored refrigerated and delivered within 2 days.26. Because intestinal cleansing agents used in colonoscopy preparation are known to significantly alter the intestinal microbiome and metabolome.63samples collected within 1 month of use were excluded from analysis. Each participant received three fecal collection tubes: one containing Cary-Blair medium (Toyobo) and two without preservatives.26. Samples preserved in Cary-Blair medium were used for gut microbiome analysis, as this medium stabilizes microbes…
Gn Health
