Hybrid-sport participation is associated with gut microbiota composition: an exploratory longitudinal study

Hybrid sports combine high-intensity resistance exercise with sustained aerobic demands, yet their association with gut microbiota composition and response to dietary or microbiota-targeted interventions remains unclear. Hybrid sports athletes have rarely been examined in human gut microbiome studies, which have largely focused on endurance disciplines. We investigated whether the combined study-group characteristics, including exercise modality, dietary intervention, and microbiota-targeted supplementation, were associated with differences in gut microbiota composition Thirty-eight adults were recruited into three groups: Hyrox® athletes (dietary intervention plus microbiota-targeted supplementation), Muay Thai fighters (dietary intervention), and sedentary adults (no intervention). Stool samples were collected at from all participants and at follow-up from a subset after the three-month study period. Full-length 16S rRNA gene sequencing was performed for all samples (n = 38), while paired shotgun metagenomic sequencing was conducted in 15 participants with complete paired samples. Alpha diversity did not differ significantly between study groups or over time. Beta diversity analysis identified sex as the strongest determinant of microbial community structure, whereas study group showed a modest association. Differential abundance analyses identified study-group-associated differences in selected taxa, including lower Enterococcaceae abundance in Hyrox® athletes and differences in Dialister hominis, Dialister massiliensis, and Succiniclasticum ruminis. Paired differential-abundance analyses accounting for repeated measurements identified a limited number of significant species- and family-level taxa, including subgroup-specific changes in Dialister succinatiphilus, Megasphaera elsdenii, Clostridium herbivorans, and Vampirovibrio chlorellavorus. Exploratory shotgun metagenomic analyses performed in a subset did not identify statistically significant gene- or pathway-level differences after multiple-testing correction, although descriptive differences were observed in selected pathways. Together, these findings suggest that the study-group characteristics were associated with fine-scale differences in gut microbiota composition. No significant large-scale changes in community-level microbial diversity were detected over the study period, whereas paired differential-abundance analyses in participants with complete follow-up samples identified a limited number of significant taxon-level changes. Because exercise modality, dietary intervention, and microbiota-targeted supplementation differed simultaneously between study groups, these findings should be interpreted as observational and hypothesis-generating rather than evidence of independent effects of exercise modality. Validation in larger controlled longitudinal studies is therefore required.