Advancing the Exercise-Microbiome Axis: A Methodological and Bioinformatic Roadmap from Short-Read Standards to Long-Read Frontiers and Multi-Omics Integration

PURPOSE: Exercise–microbiome research is expanding rapidly, but methodological heterogeneity and technical limitations still hinder reproducibility and mechanistic interpretation. This review provides a comprehensive methodological roadmap to overcome these barriers.METHODS: We conducted a structured literature search in PubMed, Web of Science, and Scopus for records published between 1998 and 2025 using predefined combinations of exercise and gut-microbiome terms. After deduplication, titles/abstracts and full texts were screened according to prespecified criteria, yielding 99 eligible studies on aerobic and resistance/anaerobic exercise and gut microbiota. Evidence is critically appraised across standard short-read 16S rRNA protocols, shotgun metagenomics, and emerging long-read sequencing, as well as metatranscriptomics, metabolomics, and associated bioinformatics pipelines. A PRISMA-style flow diagram summarizes the study-selection process.RESULTS: Exercise across diverse modalities reshapes gut microbial diversity and community structure, frequently enriching taxa such as <i>Akkermansia muciniphila</i> and <i>Veillonella</i> and enhancing production of short-chain fatty acids (SCFAs). SCFAs strengthen the intestinal barrier, activate anti-inflammatory pathways, and supply energy substrates for colonocytes and exercising muscle. Long-read sequencing now enables species- and strain-level resolution beyond short V-region amplicons, while inclusion of the gut mycobiome and virome expands ecological scope. Multi-omics designs integrating metagenomics, metatranscriptomics, and metabolomics connect microbial composition with functional outputs and host metabolic adaptations.CONCLUSIONS: The future of exercise–microbiome science lies not in enlarging static catalogs of responsive microorganisms but in constructing individual-level predictive models. Integrating long-read sequencing and multi-omics with standardized training metadata will enable precision exercise prescriptions and microbiome-targeted interventions, including tailored probiotics, synbiotics, and nutrition strategies. Adoption of these advanced methodologies can accelerate mechanistic insight and promote translation of exercise– microbiome research into athletic performance optimization and clinical practice.