Cold induced pain elicits reproducible breath metabolomic responses across geographically distinct populations

Observer-independent assessment of nociceptive states is particularly important in non-communicative or vulnerable patient populations, where self-report is unavailable. In this proof-of-concept study, we explored whether exhaled breath metabolomics, measured in real time using secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS), can capture rapid physiological responses to the cold pressor test (CPT), a standardized model of acute sympathetic and nociceptive activation. Forty healthy adults (n = 19 in Switzerland, n = 21 in China) underwent four breath measurements: two pre- and two post-CPT intervention. Mass spectral data revealed a reproducible and rapid metabolic shift, with over 400 upregulated features consistently observed across both cohorts. Pathway enrichment and correlation analyses implicated amino acid metabolism (e.g., glutamate, arginine, and threonine) and stress-related pathways (e.g., creatine, butanoate, and citric acid cycle intermediates). A neural network classifier distinguished pre-vs. post-CPT states (AUC = 0.856, 78% accuracy). Our findings support real-time breath analysis as a non-invasive method for detecting short-term physiological responses.