This thesis aimed to develop a running heat tolerance test (RHTT) to assess changes in heat tolerance and to investigate the mechanism and optimisation of heat acclimation (HA) for female athletes. The first study introduced a RHTT and assessed its repeatability. Results demonstrate good agreement, strong correlations and small differences between repeated trials. The typical error of measure values suggested low within-participant variability. Furthermore, the RHTT was effective in differentiating between individuals’ physiological responses, demonstrating that heat tolerance lies along a continuum. The second study examined the sensitivity of the RHTT to changes in heat tolerance and to evaluate individual responses to HA. Results demonstrate that the RHTT is sensitive to changes in heat tolerance and that the magnitude of adaptation is highly individual; supporting the use of the RHTT in future investigations. Reducing thermal strain through HA in not fully understood for a female population. The third study compared males’ and females’ temporal patterning to short-term HA (STHA; 5-d) and long-term HA (LTHA; 10-d). The RHTT was used to quantify changes in heat tolerance. The results confirm that whilst STHA may be effective in achieving partial adaptation in males and females, females require LTHA to establish reductions in thermoregulatory and cardiovascular strain. Improved thermotolerance following HA, reduces disruptions to cellular homeostasis principally, but not exclusively, by increasing basal heat shock protein 72 following transcription of its gene (Hsp 72 mRNA) as part of the heat shock response (HSR). The fourth study compared males’ and females’ Hsp72 mRNA response during STHA and LTHA. The similar transcription of Hsp72 mRNA observed in all participants suggests that there are no differences in the endogenous criteria to elicit the HSR between sexes. The fifth study assessed the effectiveness of preceding STHA with a passive heat exposure (HAsauna) in females. HAsauna resulted in reductions in thermoregulatory, cardiovascular and perceptual strain. The adaptation pathway was likely mediated in part, by plasma volume expansion and an improved thermoeffector and thermosensitivity response of the sudomotor function. Together, evidence in this thesis supports the notion that special considerations need to be taken when using HA to attenuate thermoregulatory strain in female athletes prior to training and competing in the heat.