Transport in containers and an unfamiliar environment are potent stress-inducing factors in domestic cats. This stress can be evaluated using cortisol concentration in serum and through other related physiological responses, such as increased glycaemia and body temperature. There is evidence that in some animal species, while subjected to psychological stress, lower right tympanic temperature is associated with emotional activation. In this study we evaluated the relationship between tympanic temperature, rectal temperature, and glycaemia and stress level as represented by cortisolaemia in domestic cats. Two groups of 18 cats were separated according to higher or lower cortisol serum levels after transportation and exposure to an unfamiliar environment. The right tympanic temperature was lower in the low cortisol group than in the high cortisol group. No significant differences in left tympanic temperature, rectal temperature, and glycaemia were observed between the low cortisol and high cortisol group. The relationship between emotional stress and tympanic temperature is in accordance with a possible emotional lateralisation in domestic cats. This functional tympanic membrane temperature technique could be an alternative method to evaluate the psychological stress in domestic cats.