Cortisol concentrations in hair are biomarkers of health status and seasonal change, but not urbanisation, in wolves

Wolves have reclaimed much of their former range in Europe, including areas with a high human footprint, yet the physiological impacts of human‐associated stressors remain poorly understood. We first examined methodological factors affecting hair cortisol concentrations (HCC) in wolves by using a ‘shave/re‐shave' design to assess hair growth rate and seasonal variation in four captive individuals. We also evaluated the effects of body region, collection method, and the relationship between faecal glucocorticoid metabolites (FGCMs) and HCC. Hair grew faster in summer, but HCC peaked in hair grown during winter, likely reflecting seasonal elevations in glucocorticoids (GCs) due to reproduction and varying metabolic demands. FGCMs showed comparable seasonal fluctuations and were significantly correlated with HCC. Additional samples collected by brushing the belly showed higher HCC than those from the back. To then assess the effects of urbanisation on HCC in free‐ranging wolves, we analysed hair samples from 254 carcasses from Italy and quantified human influence using the human footprint index (HFI). Contrary to our prediction, the HFI did not explain variation in HCC. This result may reflect the absence of individuals exposed to extreme urbanisation, uncertainty regarding whether carcass locations represented true home ranges, and the possibility that broad landscape indices such as the HFI fail to capture more immediate anthropogenic stressors. In contrast, health status and season significantly predicted HCC: wolves with chronic injuries or infectious diseases exhibited elevated HCC, and individuals that died in winter had higher HCC than those that died in other seasons. We discuss these results in the context of the wolf breeding season and dispersal period, and highlight the importance of considering seasonal demands, health status, and methodological constraints when interpreting GC patterns in wolves.