With the aim of assessing positive and negative emotional states in animals, behavioural and physiological reactions of sheep were observed in exemplary situations. Fourteen ewes were subjected to situations likely to induce states of negative, intermediate and positive emotional valence, respectively: separation from group members, standing in the home pen, and being voluntarily groomed by a familiar human. The latter was chosen as a positive situation since sheep approached the familiar experimenter, and animals stayed close-by while being groomed. The animals' ear postures, eye aperture, and cardio-respiratory activity were recorded over a four-minute period. Data were analysed using linear mixed-effects models. Moreover, measures were correlated to test whether they could be assessed in ten-second time periods. The proportion of axial ear postures shown by sheep increased linearly from separation from group members (model estimate: 0.05) towards standing in the home pen (0.31) and being groomed (0.58; p<0.001). Relative eye aperture followed an inverse pattern (0.81, 0.78, 0.74; p=0.02). Similarly, the values of two cardiac measures, mean inter-heartbeat interval (p<0.001) and heart-rate variability (p<0.001), decreased from separation from group members towards standing in the home pen and being groomed. Measurements made over ten-second and longer time periods correlated for behavioural and cardiac variables (Spearman rS=0.4-0.8, p<0.01). In conclusion, findings indicate that ear postures, eye aperture and cardiac measurements were useful for differentiating situations likely to induce negative, intermediate and positive emotional states in sheep. Moreover, cardiac variables can be measured in ten-second periods, allowing for the rapid detection of short-term emotional reactions.