Simple SummaryLameness is the most common cause of reduced performance in equids. Therefore, its detection, accurate diagnosis, and appropriate treatment are important for animal welfare and economics. Subjective evaluation of a lame horse by visual assessment is prone to error. To objectify the examination, several computer-based systems have been developed. While kinematic investigations focus on the detection of movement asymmetries (e.g., of head, pelvis, withers) with the help of position sensors, kinetic examinations are based on pressure measurement under the hooves. In the current study, horses with unilateral forelimb lameness were equipped with a non-invasive pressure measurement system on both forelimbs simultaneously. Bilateral vertical force distribution (in kg) was evaluated during all phases of stance (landing, midstance, breakover) before and after diagnostic anaesthesia. Vertical force was reduced on the lame limb compared to the sound limb before diagnostic anaesthesia. After positive diagnostic anaesthesia, asymmetries were neutralised: vertical force increased on the lame limb, with breakover being most affected. In conclusion, the current pressure measurement system can be used to objectify lameness examinations in a clinical setting. Both lameness and diagnostic anaesthesia influence the particular phases of stance differently. This might contribute to a better understanding of equine gait and lead to individually optimised shoes.Kinetic examinations of horses with induced lameness as well as the effect of perineural anaesthesia in sound horses have shown promise, but clinical studies regarding the effect of diagnostic anaesthesia during the different stance phases are rare. Fourteen horses with unilateral forelimb lameness were examined with the Hoof™ System during trot to assess vertical force distribution (in kg) affecting both front hooves before and after diagnostic anaesthesia during landing, midstance, and breakover. For statistical analysis, a covariance analysis with repeated measurements regarding the limb (lame/sound) as well as anaesthesia (before/after) and the covariable body weight was performed. The p-values for the pairwise comparisons were adjusted using the Bonferroni–Holm correction (p < 0.05). For all phases of the stance, a significant interaction between the factors limb and anaesthesia was shown. Before diagnostic anaesthesia, vertical force was significantly reduced on the lame limb compared to the sound limb during landing (−25%, p < 0.001), midstance (−20%, p < 0.001) and breakover (−27%, p < 0.001). After anaesthesia, the difference between both forelimbs was not significant anymore for all phases. The vertical force on the lame limb increased significantly after positive anaesthesia during the whole stance phase, with breakover being most affected (+27%, p = 0.001). Pressure measurements with the Hoof™ System can be used to evaluate the effect of diagnostic anaesthesia in a clinical setting with pain-related vertical force asymmetries being neutralised after diagnostic anaesthesia. Breakover is the main event influenced by lameness.