Purpose The physiological strain index (PSI) was developed to assess individuals' heat strain, yet evidence supporting its use to identify individuals at potential risk of reaching a thermal tolerance limit (TTL) is limited. The aim of this study was to assess whether PSI can identify individuals at risk of reaching a TTL. Methods Fifteen females and 21 males undertook a total of 136 trials, each consisting of two 40-60 minute periods of treadmill walking separated by similar to 15 minutes rest, wearing permeable or impermeable clothing, in a range of climatic conditions. Heart rate (HR), skin temperature (T-sk), rectal temperature (T-re), temperature sensation (TS) and thermal comfort (TC) were measured throughout. Various forms of the PSI-index were assessed including the original PSI, PSIfixed, adaptive-PSI (aPSI) and a version comprised of a measure of heat storage (PSIHS). Final physiological and PSI values and their rate of change (ROC) over a trial and in the last 10 minutes of a trial were compared between trials completed (C, 101 trials) and those terminated prematurely (TTL, 35 trials). Results Final PSIoriginal, PSIfixed, aPSI, PSIHS did not differ between TTL and C (p > 0.05). However, differences between TTL and C occurred in final T-sk, T-re-T-sk, TS, TC and ROC in PSIfixed, T-re, T-sk and HR (p < 0.05). Conclusion These results suggest the PSI, in the various forms, does not reliably identify individuals at imminent risk of reaching their TTL and its validity as a physiological safety index is therefore questionable. However, a physiological-perceptual strain index may provide a more valid measure.