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Vaginal examinations are routine procedures on dairy farms to diagnose pregnancy or puerperal diseases. Cows express their discomfort in certain situations with discrete behavioural reactions. These reactions can be described in their occurrence and extent. Although there is evidence that human contact is potentially stressful for cows, the impact of vaginal examinations on animal welfare has not been evaluated. Therefore, we hypothesized that (1) cows show discomfort before and during vaginal examination with different behavioural reactions, (2) these reactions can be semi-quantitatively scored and (3) the examination with a Metricheck device is less invasive than an examination with the gloved hand. In experiment 1, the behaviour of 10 cows during vaginal examination was videotaped and analysed. In total, 15 different behavioural reactions were identified. Based on these observations, a numerical rating system was created. The avoidance reactivity score (ARS) includes evasive reactions, which are scored on a 4-point scale, and signals of discomfort, scored with 1 point each. In Experiment 2, evasive reactions of cows were videotaped and scored by two observers and three times by the examiner. The inter- and intra-repeatability was 0.70 and 0.87, respectively. In experiment 3, vaginal examinations of 30 cows were videotaped and scored with the ARS by four observers and three times by one observer. The inter- and intra-observer repeatability during examination was 0.44 and 0.81, respectively. In experiment 4, 435 vaginal examinations were conducted either with the gloved hand (group GH) or the Metricheck device (group MD). Behaviour before and during examination was scored. The median ARS increased from 1 before (IQR: 1–2) to 3 during examination (IQR: 2–4) and cows in the group MD showed less avoidance reactions compared to cows in the group GH (P < 0.05). Parity, days in milk, vaginal discharge or repeated examinations did not influence the ARS. Our study provides evidence that vaginal examinations cause stress in cows. Furthermore, we demonstrated that behavioural reactions can be assessed with a score. Substantial inter-observer and substantial intra-observer repeatability proves that the ARS can be applied in practice. The ARS – although imperfect – might be a useful tool in the field and in research to estimate a cow's stress level.