How to conceptualize mixed emotional states is a central issue in the field of affective science. Nondifferentiation, additive, and emergence accounts of mixed emotions make divergent predictions regarding physiological responses in mixed emotions. To test these predictions, 43 women watched film clips that elicited amusement, disgust, or mixed emotions while feeling self-report, facial electromyography, cardiovascular, electrodermal, and respiratory measures were assessed. Simultaneous self-reports of amusement and disgust confirmed elicitation of a mixed emotional state. Physiologically, mixed emotions differed from pure amusement and pure disgust both in intensity and pattern. This suggests a distinct physiological response of the mixed emotional state, as predicted by the emergence account of mixed emotions. Implications for emotion theory and research are discussed.