Exploring Sweat Protein Composition as a Biomarker for Heat Acclimation in Men and Women

Purpose: To test the hypothesis that heat acclimation (HA) induces sex-specific and stress-specific shifts in sweat-associated protein abundances and to identify potential sweat biomarkers of thermal adaptation. Methods: Thirty-two physically active adults (17 females, 15 males; 22.9 ± 3.6 years, VO 2 max 47.4 ± 7.0 mL·kg - 1 ·min - 1 ) completed an eight-day HA protocol involving treadmill walking and running in a controlled heat chamber (40°C, 40% relative humidity). Sweat samples were collected from the thigh during pre-HA (HE1) and post-HA (HE8) sessions using gauze confined with Tegaderm. Sweat samples (~30 µg protein) were alkylated, acidified, and subjected to S-Trap digestion with porcine trypsin. Peptides were analyzed via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with label-free quantitation. Differential protein expression and pathway analyses were performed using Scaffold and R, with statistical significance set at p < 0.05 and p<0.01. Physiological measures (rectal temperature, heart rate, sweat rate) and perceptual responses (rating of perceived exertion) were recorded. Results: Physiological adaptations included significant reductions (p<0.05) in rectal temperature (-0.42°C), heart rate (-23 bpm), and perceived exertion (-2.2) at HE8 compared to HE1, confirming HA. Proteomic analysis identified 911 proteins, with 223 unique proteins showing significant change abundance changes (p < 0.05). HE1 proteomics highlighted acute stress responses, such as extracellular matrix remodeling and immune defense activation, while HE8 proteomics reflected enhanced keratinocyte differentiation, intermediate filament organization, and tissue-specific adaptations. Sex-specific differences included greater enrichment of wound-healing and antimicrobial proteins in males and increased metabolic regulation and protein turnover in females. Notable proteins included THSD4, S100-P, CEACAM5, and KLK11, which exhibited significant shifts associated with cellular protection, structural adaptation, and immune modulation. Conclusion: HA induces distinct shifts in sweat proteomics, transitioning from acute stress responses to specialized thermoregulatory adaptations. Sex differences in proteomic expression provide insights into individualized thermal adaptation mechanisms. These findings highlight the potential of sweat as a biofluid for non-invasive monitoring of HA and support the discovery of biomarkers for thermotolerance and physiological resilience. Future work should validate these findings across diverse populations and integrate multi-biofluid approaches. Funding: DoD BA200299 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.