BACKGROUND: Chronic fatigue syndrome (CFS) is a crippling illness with limited effective treatment options. Recent findings have suggested that ginseng stem and leaf saponins (GSLS) possess anti-fatigue effects. However, the detailed mechanisms by which GSLS impact CFS have yet to be fully elucidated. PURPOSE: This study aimed to explore the efficacy and underlying mechanisms of GSLS in the treatment of peripheral and central fatigue in mice with CFS. METHODS: A CFS model was established in male C57BL/6 mice, which were then subjected to prolonged swimming tests. The validity of this model and the efficacy of GSLS were verified using the forced exhaustion swimming test and the Morris water maze test. The anti-fatigue mechanism of GSLS was verified using pathological section observations, biochemical index tests, neurotransmitter analysis, and Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-MS) metabolomics analysis. RESULTS: GSLS prolonged the swim time-to-exhaustion in CFS mice and reduced escape latency in the water maze, revealing their anti-fatigue and memory-improving effects. GSLS significantly protected both peripheral (liver) and central (hippocampus) tissues, as shown in pathological sections. Biochemical indices suggested that GSLS mitigate peripheral fatigue by reducing energy expenditure and oxidative damage and inhibiting the production of harmful metabolites. Furthermore, GSLS altered the abnormal levels of 14 neurotransmitters in the brains of CFS mice, including seven upregulated and seven downregulated neurotransmitters, highlighting their role in mitigating central fatigue through neurotransmitter regulation. Serum metabolomics analysis identified alterations in 19 metabolites across 11 metabolic pathways, thereby elucidating the potential mechanisms by which GSLS treat CFS. CONCLUSION: GSLS ameliorate damage caused by both peripheral and central fatigue. They also act on multiple pathogenic mechanisms by modulating neurotransmitters and serum biomarkers, making them a potential therapeutic agent for CFS.