**5. Conclusions**

The ability of plasma, feces and hair to provide multi-temporal information about physiological state proved to be very useful in the present study. Although the use of di fferent biological samples increased the di fficulty in the interpretation of our results, it allowed a broader panorama, which was more complete and reliable to understand given such a complex topic like stress reactions. We found evidence that repeated exposure of our red deer population to game hunting activity had an impact on stress levels, which can have important consequences for sustainability and conservation of this species. Specifically, stress can a ffect population dynamics, by changing foraging and breeding behavior, animal welfare, and, ultimately, the evolutionary processes, by changing individual fitness and selection [2,5,6]. Thus, exploring these topics is crucial to understanding the implications of hunting for the conservation of this species and to improve hunting managemen<sup>t</sup> activities. Our study highlights the fundamental and broad role of stress in wildlife, emphasizing the need for more studies capable of clarifying how di fferent biological matrices may be useful to evaluate the impacts of human pressure on wildlife, both in terms of stress level and stress processes.

**Author Contributions:** S.V., J.A. and A.A.d.S. conceived the experiments and performed the field work. S.V. and R.P. performed the laboratory analysis. J.A. and A.A.d.S. performed data analysis. S.V. and J.A. drafted the manuscript. S.V., A.A.d.S., R.P., K.E.R., J.P.S. and J.A. contributed to the writing, discussion and revision of the manuscript and have read and approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by POPH/FSE funds from the Portuguese Foundation for Science and Technology (FCT) through the fellowship of J. Alves (SFRH/BPD/123087/2016) and A. Alves da Silva (SFRH/BD/75018/2010). This study was funded by Instituto do Ambiente, Tecnologia e Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra (IATV-UC) and supported by FCT—Fundação para a Ciência e a Tecnologia, I.P. within the project UID/BIA/04004/2019.

**Acknowledgments:** We thank the hunting areas managers of the ZCM of Lousã, ZCM of Vila Nova and ZCA of Miranda do Corvo for the collaboration during the collection of the biological samples. We also thank Filipe Rocha, Luis Pascoal da Silva and Fernanda Garcia for their help in the collection and preparation of the biological samples, Raquel Alves for helping with the classification of blood cells, and Marlon Millard for performing the EIA analyses.

**Conflicts of Interest:** The authors declare no conflicts of interest.
