Tehran University of Medical Sciences Traditional and Integrative Medicine 2476-5104 11 2 2026 06 28 2128 2128 Afsaneh Oskouie Department of Basic Sciences, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran Hossein Soraki Aliabad Traditional Medicine and Materia Medica Research Center and Department of Traditional Medicine, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Rasool Choopani Zanjani Traditional Medicine and Materia Medica Research Center and Department of Traditional Medicine, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Sajjad Sadeghi Traditional Medicine and Materia Medica Research Center and Department of Traditional Medicine, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Reyhaneh Yekta Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Shahpar Kaveh Traditional Medicine and Materia Medica Research Center and Department of Traditional Medicine, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Mohammad Fayaz Allameh Tabataba'i University, Tehran, Iran Zeinab Karimi Tuberculosis and Lung Diseases Research Center, Illam University of Medical Sciences, Illam, Iran Alireza Rahmani Student Research Center, Iran University of Medical Sciences, Tehran, Iran 2025 05 15 2025 11 14 Persian Medicine (PM) categorizes individuals into distinct temperaments (Mizaj) based on their unique morphological, physiological, and psychological characteristics. However, the scientific community requires further evidence to validate these temperament classifications. This study aimed to compare the serum metabolite profiles of healthy individuals with hot-wet and cold-dry temperaments using liquid chromatography-mass spectrometry (LC-MS). A descriptive cross-sectional untargeted metabolomics investigation was conducted between 2022-2023, focusing on 20-25-year-old male participants exhibiting hot-wet and cold-dry temperaments. Blood samples were collected, and their serum metabolite profiles were analyzed using LC-MS. The obtained data were then compared between the two groups, using HMDB, METLIN, KEGG databases, and MetaboAnalyst web-based platform for pathway analysis. Among the 59 differentially expressed metabolites, 56 (94.9%) exhibited significantly higher abundance in individuals with hot-wet temperament compared to those with cold-dry temperament (fold-change ≥1.5, p < 0.05, FDR-corrected q < 0.05, VIP >1.0). Enrichment analysis based on KEGG revealed seven metabolic pathways with higher expression in the hot-wet temperament group compared to the cold-dry group, including the alanine, aspartate, and glutamate metabolic pathways, purine metabolism, arginine and proline metabolism, aminoacyl tRNA biosynthesis, citrate cycle (TCA cycle), the pentose phosphate pathway, and glycolysis and gluconeogenesis. This preliminary study highlights significant differences in the serum metabolic profiles of individuals with hot-wet temperament compared to those with cold-dry temperament, particularly in protein and energy metabolism pathways. These findings provide preliminary biochemical insights into the concept of temperament classification within PM. Further research involving larger and more diverse populations is needed to confirm and expand upon these observations. https://jtim.tums.ac.ir/index.php/jtim/article/view/2128