Wildlife tourism is increasing worldwide and monitoring the impact of tourism on wild populations is of the utmost importance for species conservation. The Magellanic penguin Spheniscus magellanicus colony at Martillo Island, Argentina, was studied in the 2016–2020 breeding seasons. In all seasons, adults and chicks belonged to: (i) an area close to or within the tourist trail or (ii) an area far from the tourist trail and out of sight of the tourists. Blood samples were taken for carbon and nitrogen stable isotope composition, in order to estimate trophic niches, and for smears that were made in situ and were then stained in the laboratory where leucocyte counts and differentiation were made under optical microscope. Heterophil to lymphocyte ratios were used as proxies of stress. Repeated sampling showed individual stress levels reduced while wintering. In 2017, stress levels and trophic values were lower than 2018 for the same individuals. Trophic levels did not differ between tourism and no tourism areas within each season, and differed between 2017 and the remaining seasons, indicating a possible diet shift that year. Stress levels were higher for the tourism area than the no tourism area for adults and chicks in all years except for 2020, when stress levels in the tourism area were lower and similar to the no tourism area that year and previous years. Vessel transit within the Beagle Channel and tourist visitation to the penguin colony was greatly reduced in 2020 due to the Covid-19 pandemic. A combination of internal characteristics and external factors may be affecting the stress physiology of individuals. Therefore, future research should include sampling of multiple aspects of penguin physiology, behaviour and environmental context in order to evaluate each effect on Magellanic penguin stress and, ultimately, inform the conservation of this iconic species in time.