Profiling the Athletes’ Gut Microbiome: A Critical Methodological Perspective on 16S Metabarcoding and Shotgun Metagenomics

The growing interest in the role of the gut microbiome in athletic performance has led to the application of various sequencing technologies in this field. This review critically examines the sequencing methodologies used in microbiome studies on physical performance and sport, comparing their advantages, limitations, and applicability. In particular, the focus is on 16S metabarcoding and shotgun metagenomics, evaluating how these methodological approaches influence the interpretation of results in sports contexts. Close attention is directed toward technical challenges, methodological biases, and future perspectives, including emerging technologies and multi-omics approaches. This review aims to bridge the gap between methodological rigor and sports-specific applicability, providing evidence-based methodological guidance to support researchers in designing robust athlete microbiome studies and translating sequencing-derived findings into concrete applications for performance and sports health.