Heat stress from high temperatures has been recognised as a threat to several bird species and one that is likely to increase in severity and frequency as a result of global environmental change. Many seabirds are particularly vulnerable as their adaptations to forage in cold water can make it hard for them to resist heat gain while on land. The African Penguin Spheniscus demersus forages in cool water (10–20°C) but breeds and moults on land where temperatures can exceed 30°C. Little is known about its thermal physiology and when heat stress will occur. Here, we provide the first account of changes in body temperature and behaviour in African Penguins in response to an experimental increase in ambient temperature from 20°C to 35°C. Surprisingly, the highest body temperature observed (39.2°C) was recorded at the start of the study. Presumably, this response in body temperature was due to the stress of transport and handling. Penguins returned to normal body temperature (37.3°C) after 3.5 hours and maintained normothermia for roughly an hour. We then observed the onset of heat storage at an ambient temperature of 29°C, whereafter body temperature increased steadily at a rate of ca.0.2°C per 1°C increase in ambient temperature. Panting commenced at an ambient temperature of 31.4°C, when body temperature was 37.8°C. In addition to panting, penguins changed their posture to facilitate heat dissipation by standing, raising their heads, and extending their flippers. Our results corroborate field observations and support the argument that African Penguins are highly vulnerable to heat stress in the near future as extreme heat events become more severe and frequent due to climate change. Our results also confirm that penguins are sensitive to handling, which elicits a hyperthermic response. Given the general sensitivity of penguins to disturbance, from a conservation perspective, we advise that cautionary measures be implemented at colonies during critical life-history stages.