Athlete or Season? Gut Microbiota Variance in Elite Volleyball Players: A Compositional Performance-Based Reanalysis

Background: The gut microbiota is an emerging factor in athletic performance. Methodological limitations persist in longitudinal microbiome studies, particularly regarding the compositional nature of microbiota data and the lack of standardized performance metrics in team sports. The present study applies a Compositional Data Analysis (CoDA) framework with ANOVA-Simultaneous Component Analysis (ASCA) to investigate longitudinal gut microbiota dynamics in elite volleyball athletes and introduces a novel Technical Performance Level (TPL) metric for objective performance quantification. Methods: Seven elite male volleyball athletes from the Italian SuperLega Championship were monitored across four sampling timepoints (T0, T1, T2, T3) during the Regular Season, Rest Period, and Tournament Period. Fecal samples underwent 16S rRNA metabarcoding. CLR-transformed data were analyzed at the Phylum, Family, and Genus level using ASCA with Season, Player, and their interaction. Statistical significance was assessed using the Freedman-Lane permutation test (10,000 iterations). Within-subject associations between TPL and CLR-transformed microbial features were assessed by repeated-measures correlation. Results: Season and Player effects were statistically significant at all taxonomic levels (p ≤ 0.0004), whereas the Season × Player interaction was significant at the Family and Genus levels (p ≤ 0.002) but not at the Phylum level (p = 0.108). The Player effect represented the largest source of variance at the Family (51.31%) and Genus (50.75%) levels. At the Phylum level, the Season × Player interaction accounted for the largest share of variance (36.23%), although it did not reach significance. Within-subject correlation analyses revealed no statistically significant association between microbial features and TPL at any taxonomic level, and none remained significant after correction for multiple comparisons. Conclusions: The application of a compositional data analysis framework modifies the interpretation of gut microbiota dynamics in elite volleyball athletes compared to standard approaches. Individual athlete identity accounted for most of the compositional variance in the microbiota, whereas exploratory within-subject analyses did not reveal statistically significant associations between taxa and TPL. Given the small cohort, any observed trends should be interpreted as exploratory and hypothesis-generating until confirmed in adequately powered cohorts. Longitudinal monitoring of gut microbiota could support individualized surveillance of gut health in elite sport.