Simple SummaryHorse training exposes horses to many difficult learning challenges, including tasks that run counter to their natural behaviour and research from neuroscience can be used to understand how horses respond to these learning challenges. Horses can also quickly develop habits, however if the habit is undesirable, the horse may be subjected to retraining where it must learn to suppress the unwanted behaviour and learn a new response. This is called cognitive flexibility and is underpinned by complex neural processes and can be impaired by exposure to repeated or chronic stress. We trained horses to acquire habit-like responses in one of two industry-style aversive instrumental learning scenarios (moving away from the stimulus-instinctual-IS or moving towards the stimulus-non-instinctual-NIS) and evaluated the effect of repeated stress exposures on their cognitive flexibility in a reversal task. We measured heart rate, salivary cortisol and serum brain derived neurotrophic factor to infer possible neural correlates of the learning outcomes. Horses trained in the NIS task took longer to learn task than the horses trained in the IS task, however they were quicker to learn the IS task during the reversal. There was no significant effect of the stress exposures on cognitive flexibility, however the stress did make horses more sensitive to the aversive training stimuli. These results provide guidance for trainers to adapt their practices when training tasks that are difficult for horses to learn and during retraining. In particular, even when horses have been trained to approach aversive stimuli they can quickly learn to avoid them. This has implications for retraining unwanted responses during aversive situations such as trailer loading.Horse training exposes horses to an array of cognitive and ethological challenges. Horses are routinely required to perform behaviours that are not aligned to aspects of their ethology, which may delay learning. While horses readily form habits during training, not all of these responses are considered desirable, resulting in the horse being subject to retraining. This is a form of cognitive flexibility and is critical to the extinction of habits and the learning of new responses. It is underpinned by complex neural processes which can be impaired by chronic or repeated stress. Domestic horses may be repeatedly exposed to multiples stressors. The potential contribution of stress impairments of cognitive flexibility to apparent training failures is not well understood, however research from neuroscience can be used to understand horses’ responses to training. We trained horses to acquire habit-like responses in one of two industry-style aversive instrumental learning scenarios (moving away from the stimulus-instinctual or moving towards the stimulus-non-instinctual) and evaluated the effect of repeated stress exposures on their cognitive flexibility in a reversal task. We measured heart rate as a proxy for noradrenaline release, salivary cortisol and serum Brain Derived Neurotrophic Factor (BDNF) to infer possible neural correlates of the learning outcomes. The instinctual task which aligned with innate equine escape responses to aversive stimuli was acquired significantly faster than the non-instinctual task during both learning phases, however contrary to expectations, the repeated stress exposure did not impair the reversal learning. We report a preliminary finding that serum BDNF and salivary cortisol concentrations in horses are positively correlated. The ethological salience of training tasks and cognitive flexibility learning can significantly affect learning in horses and trainers should adapt their practices where such tasks challenge innate equine behaviour.