Animal welfare is a result of accumulated negative and positive emotions. Therefore, it is important to limit the former and promote the latter. Doing so requires a precise identification of these emotions. The aim of this study was to characterize the behaviours, facial expressions and locomotor parameters of 21 horses in two conditions with opposite valence, presumed to induce positive anticipation and frustration. In the positive anticipation situation, the horse was led to a bucket of food, knowing that they would be allowed to eat it. In the frustration situation, experimenters indicated to the horse that food was available without allowing them to eat it. In the positive anticipation situation, horses exhibited a lower neck position with the ears forward and upper lip advanced and went faster by increasing their stride frequency accompanied by increased global locomotor activity. In the frustration situation, horses exhibited a higher neck position with the ears backward or to the side, accompanied by ear movements and eye blinks, and interacted more with the experimenters. This study describes new possible indicators of positive anticipations and frustration in horses.