Introduction: Short-term heat acclimation (STHA) with no fluid intake, initiates physiological adaptations that has been shown to improve heat tolerance and human performance. Heat shock protein 70 (HSP70) is a highly inducible stress protein elevated in response to heat stress, as well as other conditions such as exercise, cellular acidosis and hypoxia. In male participants, it has been reported that after bouts of STHA, there is an increase in basal intracellular HSP70 expression and an attenuation of the HSP70 response to a subsequent heat stress. HSP70 response could be used as a molecular marker of heat adaptation. However, limited data exists on heat shock protein response in female participants. Therefore, the aim of this work was to determine the effect of STHA on HSP70 in a trained female and male cohort. Methods: Seventeen participants; six females (Mean [SD]; 23 [3] y; stature 164.9 [7.0] cm; body mass 61.0 [9.2] kg; VO² max 44.01 [8.94] mL.kg-1.min-1) and eleven males (Mean [SD]; 36 [15] y; stature 175.7 [4.5] cm; body mass 79.4 [11.7] kg; VO² max 52.40 [11.01] mL.kg-1.min-1) participated in a STHA programme. This consisted of 90 minutes of heat exposure, with permissive dehydration during heat acclimation (39.5oC and 60% relative humidity), using the controlled-hyperthermia technique (~rectal temperature [Tre] 38.5oC), for five consecutive days, in females and four days in males. On the first and final day of acclimation venous blood samples were taken from the median cubital vein prior to, and immediately, after acclimation. Serum samples were analysed using a HSP70 high-sensitivity ELISA (Abcam; ab133061). HSP70 data was corrected for total protein (TP), with the no-fluid intake regime during STHA. Differences pre- and post- STHA, on the first and final day were analysed using a Wilcoxon Signed Rank test. Data are median [Q1-Q3]. Results: In females, HSP70/TP increased from 0.092 [0.088-0.093] ng/ml pre-acclimation to 0.539 [0.535-0.542] ng/ml post-acclimation (P=0.03), on day one of STHA. Similarly, on day 5 of acclimation, HSP70/TP increased from 0.096 [0.092-0.097] ng/mL pre-acclimation to 0.576 [0.558-0.584] ng/mL post-acclimation (P=0.03). The magnitude of change in HSP70/TP was no different on the first and last day of heat acclimation (P=0.16).  In males, HSP70/TP increased from 0.089 [0.085-0.092] ng/mL to 0.565 [0.549-0.582] ng/mL on day one of heat acclimation (P<0.001). On the final day of acclimation, baseline HSP70/TP decreased to 0.078 [0.077-0.082] ng/mL, compared to day 1 baseline levels (P<0.001). Interestingly, whilst the increase post-acclimation (0.506 [0.481-0.531] ng/mL), remained (P<0.001), the magnitude of change was less on the final day compared to the first day of acclimation (P=0.002). Conclusion: – Our work suggests that STHA attenuates the heat shock protein response in the male cohort only. Given the limited information regarding the heat shock protein response in females, further research is required to ascertain the kinetics of the extracellular HSP70 response in this population. Furthermore, our data suggests if HSP70 is to be used as a biomarker for heat acclimation, more research is required into the heat shock response in females.