Equine headshaking syndrome (EHS) is associated with severe headshaking in the absence of an identified external stimulus, which affects welfare of the horse as well as handling and especially rideability. Since EHS mainly occurs under the rider, the aim of this study was to investigate different effects in standardised riding tests. Therefore, riding tests (n = 227) were carried out on 16 horses affected by idiopathic EHS and 12 corresponding control horses in order to compare heart rate, saliva samples, thermographic images as well as rein tension and ethological indicators between the two groups. Statistical analysis was performed using generalised linear mixed models for (1) the heart rate, (2) the rein tension in Newton as well as (3) behaviour based on binary data structure. In particular, the study tested for the presence of headshaking (PoHS) (0 = negative, 1 = positive) and gait (−1: backwards, 0: halt, 1: walk, 2: trot, 3: canter) as well as other fixed effects depending on the model. The saliva samples as well as the thermographic images were evaluated using a mixed model. The heart rate dataset revealed that PoHS has no significant impact on the heart rate (P = 0.58); whereas, it is significantly influenced by gait (P ≤ 0.0001). The rein tension dataset indicated that this is not influenced by PoHS, but a meaningful influence was caused by the equipment used (saddle: P ≤ 0.0001) and environmental influences (wind: P ≤ 0.0001). The results of the behavioural parameters indicate that there is no difference between headshakers and control horses in terms of behaviours that indicate a negative emotional state. No difference was found between headshakers and control horses in the evaluation of saliva samples and thermographic images. Wearing a nose cover significantly reduced headshaking symptoms (head flicking: P ≤ 0.05). In summary, no connection of PoHS with the heart rate, increased cortisol levels and thermographic images as well as rein tension could be proven. Thus, it can be concluded that riding has a negligible effect on EHS. Using a nose cover leads to a reduction in frequency of headshaking movements. This study is a first step to investigate the influence of riding on horses affected by EHS and, in general, contributes to a better understanding of EHS.