The heat shock protein (HSP) response is critical to the cellular stress response. Circulating, extracellular stress‐inducible HSP72 is known to respond to exercise‐heat stress, but little is known about other, constitutively expressed and fundamental members of the HSP family. The aim of this study was to investigate the extracellular HSP27, HSP60, and HSP90α response during a prolonged cycling event in hot and humid conditions. Seventy‐six subjects (mean±SD: age, 51.64±10.47 years; height, 176.44±6.85 cm; mass, 86.78±13.93 kg) were recruited at the Hotter'n Hell 100 mile cycling event in Wichita Falls, Texas. Blood and urine samples, as well as data on body weight and gastrointestinal temperature (TGI) were recorded before (pre) and after (post) the event. Plasma was isolated from blood samples, stored at −80°C, and later analyzed with StressMarq Biosciences ELISAs for HSP27, HSP60, and HSP90α concentrations. Student's t‐test was used to test for significant differences of means. TGI significantly increased from pre to post (pre: 37.14±0.55 °C; post: 38.12±0.79 °C; p<0.05). HSP27 and HSP90α were both significantly elevated post race (pre27: 2.73±0.51 ng × mL−1; post27: 3.46±0.73 ng × mL−1; pre90α: 18.03±3.27 ng × mL−1; post90α: 21.53±4.84 ng × mL−1; p < 0.05). However, there was no significant difference in HSP60 plasma concentration from pre to post (pre: 44.05±7.38; post: 43.65±6.81; p=0.77). In conclusion, significant increases in circulating HSP27 and HSP90α suggest that chaperones required for thermotolerance and protein degradation or folding during heat stress increase in extracellular compartments post exercise‐heat stress. However, our data suggest that chaperones specifically involved in mitochondrial protein assembly or molecular danger signals like HSP60 are not elevated in blood fractions. Ongoing work will characterize cellular expression of these proteins in circulating peripheral blood mononuclear cells.Support or Funding InformationNew investigator start‐up, McNair Scholar ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.