In light of the increasing amount of in vivo human research being conducted into the expression and potential roles of extracellular heat shock protein 72 ( eHsp72), the aims of this thesis were to address a number of unanswered important methodological questions that researchers should be aware of when planning such investigations. The specific aims were; l) to examine the effect of different blood handling procedures upon eHsp72 concentration, 2) to investigate the stability of plasma eHsp72 concentration over a 24-hour period, 3) to explore the effect of acute psychological stress upon eHsp72 concentration, and 4) to investigate the utility of salivary eHsp72 as a marker of plasma Hsp72 during exercise and to speculate upon the role of sympathetic/parasympathetic stimulated release of salivary Hsp72. The assayed concentration of eHsp72 is affected by the matrix in which it is tested. Blood samples drawn in the presence of an anticoagulant (plasma) returned considerably higher concentrations of eHsp72 than samples that were allowed to clot (serum). Whilst this has obvious benefits in maximising eHsp72 detection, it has also prompted speculation that eHsp72 may have biological roles during the clotting process, since the majority of Hsp72 was unrecovered in serum samples. Acute psychological stress did not alter the concentration of eHsp72. Therefore, acute anxiety associated with participation in research studies is unlikely to affect baseline or intervention data. Additionally, it has been demonstrated that resting eHsp72 concentrations do not show a circadian variation and remain remarkably stable on a day-to-day basis. Finally, the effects of acute exercise upon sali vary and plasma eHsp72 concentration was investigated. Exercise increased plasma eHsp72, but did not significantly alter salivary eHsp72 concentration or secretion rate. Furthermore, salivary eHsp72 concentration d id not track plasma eHsp72. By utilising a caffeine-evoked sympathetic stimulation model, and measurement of salivary a -amylase and total protein, we have suggested that salivary eHsp72 is released independently of adrenergic-stimulation. To conclude, it is apparent that time of day effects and acute psychological stress do not alter eHsp72 concentration, so should not be a primary concern for future investigations into the biological roles of eHsp72. Furthermore, whilst Hsp72 in present in saliva, concentrations derived from this matrix should not be used as a proxy measure of circulating Hsp72 concentration. Finally, to maximise detection and Hsp72 recovery, blood samples should be collected in the presence of an anticoagulant.