Simple Summary How animals perceive animal-assisted interventions (AAI) has been of concern lately, especially as these activities involve many tactile stimulations. It has been shown that tactile reactivity in horses could vary greatly between individuals and could depend upon a variety of factors. Repeated tactile stimulations, according to the associated valence, may lead to lower or higher reactivity. Here we hypothesized that the numerous tactile actions, sometimes with atypical gestures, during AAI may lead to tactile sensitization. In order to test this hypothesis, we tested, with von Frey filaments, the tactile reactivity of 60 horses involved in equine-assisted interventions (EAI), conventional riding school (RS) or mixed activities (EAI-RS). The results indicate that EAI horses showed a higher tactile reactivity than EAI-RS and RS horses, showing a higher number of reactions and a higher reactivity towards thin filaments. These differences could be related to human actions during EAI, as observations of brushing sequences by participants with or without mental and/or developmental disorders revealed differences in the distribution and modalities of tactile actions: participants diagnosed with such disorders brushed more the hindquarters and showed more fragmented actions. These results call for attention towards procedures during EAI and for promoting appropriate tactile actions from participants. Tactile perception in humans varies between individuals and could depend on extrinsic factors such as working activity. In animals, there is no study relating the influence of animals' work and their tactile reactivity per se. We investigated horses' tactile reactivity using von Frey filament in different body areas and compared horses working only in equine-assisted interventions (EAI), in riding school (RS) lessons, and in both activities (EAI-RS). We further compared tactile actions by people with or without mental and/or developmental disorders during brushing sessions. The results indicated that EAI horses showed higher tactile reactivity compared to EAI-RS and RS horses, both in terms of number of reactions overall, and especially when the test involved thin filaments. All horses showed high tactile reactivity when tested on the stifle, and this was particularly true for EAI horses. These differences could be related to humans' actions, as participants diagnosed with disorders brushed more the hindquarters and showed more fragmented actions. This study opens new lines of thought on the influence of EAI working activity on horses' tactile reactivity, and hence, on horses' sensory perception. Tactile reactivity outside work, may be directly (via tactile stimulations) or indirectly (via the welfare state), influenced by working conditions.