Under controlled laboratory conditions, neurobehavioral assays such as the Psychomotor Vigilance Task (PVT) are sensitive to increasing levels of fatigue, and in general, tend to correlate with subjective ratings. However, laboratory studies specifically curtail physical activity, potentially limiting the applicability of such findings to field settings that involve physical work. In addition, laboratory studies typically involve healthy young male participants that are not always representative of a typical working population. In order to determine whether these findings extend to field-like conditions, we put 88 Australian volunteer firefighters through a multi-day firefighting simulation. Participants were required to perform real-world physical and cognitive tasks under conditions of elevated temperature and moderate sleep restriction. We aimed to examine changes in fatigue in an effort to determine the optimum objective and subjective measures. Objective and subjective tests were sensitive to fatigue outside laboratory conditions. The PVT was the most sensitive assay of objective fatigue, with the Samn-Perelli fatigue scale the most sensitive of the subjective measures. The Samn-Perilli fatigue scale correlated best with PVT performance, but explained a small amount of variance. Although the Samn-Perelli scale can be easily administered in the field, the wide range of individual variance limits its efficacy as a once-off assessment tool. Rather, fatigue measures should be applied as a component of a broader fatigue risk management system. Findings provide firefighting agencies, and other occupations involving physical work, guidance as to the most sensitive and specific measures for assessing fatigue in their personnel.