Hendra virus has caused periodic fatal disease in 90 horses and 7 humans in Australia since 1994. Epidemiological studies suggest that flying-foxes, in particular the Black flying-fox (Pteropus alecto) and the closely related Spectacled flying-fox (P. conspicillatus) are the natural reservoir hosts. This study investigates the hypothesised causal relationship between sub-optimal nutrition and physiological stress with Hendra virus infection in the Black flying-fox. During a twelve month study of flying-foxes in South East Queensland, samples were collected from captured wild Black flying-foxes for hematologic, biochemical and urine analysis, establishing normal reference ranges. The population demonstrated no significant deviation from normal hematologic or biochemical ranges across the study, but did demonstrate statistically significant temporal changes consistent with life cycle events. Measurements from Hendra virus positive animals were within the established normal ranges, but demonstrated statistically significant differences compared with Hendra virus negative animals, notably, increased lymphocyte %, decreased neutrophil %, decreased plasma triglyceride and increased plasma alkaline phosphatase levels and increased urinary protein levels. In order to investigate physiological stress, population urinary cortisol and Hendra virus urinary excretion prevalence was measured from mixed species roosts of flying-foxes from 2 geographically distinct sites (inland subtropical SE Queensland and coastal tropical Far North Queensland). This study demonstrated no significant seasonal fluctuations in Hendra virus excretion prevalence, nor urinary cortisol for the Far North Queensland population, but significant seasonal fluctuations in both Hendra virus excretion prevalence and urinary cortisol for the South East Queensland population. As population measurements are difficult to interpret due to the presence of multiple species and cohorts, a novel urinary collection and molecular analysis method was employed over a 1 year study to collect individual animal urines from the South East Queensland roost site to identify sex and species cohorts, and assess cortisol levels and Hendra virus excretion status.