Background & AimsLimited understanding exists regarding the characteristics and biological significance of the salivary microbiome in healthy individuals experiencing physiological fatigue. This study aimed to delineate the structural and functional alterations in the salivary microbiome of healthy individuals undergoing physiological fatigue compared to energetic controls, and to explore its potential as a biomarker for fatigue status.MethodsA cohort of 7 healthy individuals experiencing acute physiological fatigue (induced by prolonged study and confirmed via electroencephalography; Fatigue group, FTG) and 63 energetic healthy controls (Energetic group, ENG) were enrolled. Saliva samples were collected, from which microbial DNA was extracted. The V3–V4 hypervariable region of the 16S rRNA gene was subsequently sequenced using high-throughput technology. Bioinformatics analyses encompassed assessment of alpha and beta diversity, identification of differential taxa using Linear discriminant analysis Effect Size (LEfSe) with multi-method cross-validation, construction of microbial co-occurrence networks, and screening of fatigue-associated biomarker genera via the Boruta-SHAP algorithm. Microbial community phenotypes and potential functional pathways were predicted using BugBase and PICRUSt2, respectively.ResultsThe FTG group exhibited significantly diminished alpha diversity (Simpson index, p=0.01071) relative to the ENG group. Beta diversity analysis demonstrated significant dissimilarities in microbial community structure between the groups (p