Personal Urinary Metabotypes and Sex Differences Dominate Seasonal and Dietary Effects in a Four-Season Longitudinal Urine Metabolomics Study

Urinary metabolomics captures integrated information on host physiology, diet, and microbial co-metabolism. However, the relative contribution of stable biological traits, seasonal variation, and short-term dietary perturbations remains difficult to disentangle. This study aimed to define the hierarchy of factors shaping urinary metabolomic variability in a longitudinal four-season dietary intervention.

A total of 93 urine samples from eight healthy participants were collected across four seasons before and after a short-term controlled dietary intervention. The final dataset comprised 827 metabolomic features (levels 1–3 annotations) and nine clinical urinary variables. Global variance partitioning was assessed using single-factor PERMANOVA, while Principal Component Analysis (PCA) and cross-validated Partial Least Squares Discriminant Analysis (PLS-DA) with permutation testing were used to evaluate group separation and classification performance.

Participant identity was the dominant source of metabolomic variation, explaining 36.5% of the total variance (p = 0.005). Sex emerged as the second most important factor, accounting for 9.0% of the variance. PCA revealed clear separation between male and female samples, and PLS-DA further supported strong sex-related metabolic differences (balanced accuracy = 1.00; permutation p = 0). In contrast, season explained only 4.6% of the variance (p = 0.03), while dietary phase and pre/post-intervention status accounted for 2.6% and 1.2%, respectively, with limited evidence of global clustering. Repeated samples from the same participant were consistently more similar than samples from different individuals, supporting the existence of stable personal urinary metabotypes.

These findings indicate that individual metabolic signatures and sex-specific differences are the primary determinants of urinary metabolomic structure, whereas seasonal and dietary influences produce comparatively modest within-individual shifts. The results highlight the importance of incorporating both subject identity and sex into the design and interpretation of nutritional metabolomics studies.