Background: Many professional violin players develop pain complaints in neck and shoulder regions as a result of prolonged practices containing highly repetitive motions and awkward postures. These complaints are often diagnosed as overuse syndrome, where physical changes are not apparent and of which the etiology is not yet understood. Mechanical overload might be part of the equation. Only limited research has been done to evaluate whether the biomechanics of violin playing differ between violinists with and without overuse syndrome. Muscle activity has been reported to differ between violinists with and without pain, but findings were often inconsistent. This study aims to evaluate the biomechanics of the musculoskeletal system in the neck and left shoulder region in violin players with and without pain complaints while playing different musical excerpts. Method: Twenty violinists, 10 with and 10 without pain complaints, were playing three musical pieces containing different wrist and bow motions. These were measured using motion capture and chin-violin force measurements. An upper body model was created by combining a neck model with a shoulder model in OpenSim, which was then used to calculate joint angles and moments of the neck and left shoulder. Differences in the kinematics and kinetics of violin playing were assessed by investigating mean values as well as the variability around these average values. The mean values of the joint angles and joint moments were compared over time during selections of bowing cycles and cycles of wrist movement. The kinematic variability was assessed with the standard deviations of the mean in each playing condition. Findings: Kinematic and kinetic differences were found between symptomatic and pain-free violinists during fast bowing motions. The axial rotation of the glenohumeral joint was significantly more external in violinists with overuse syndrome compared to controls. Additionally, joint moments in several degrees of freedom of the scapulothoracic and glenohumeral joints were larger in the symptomatic group compared to the asymptomatic group. Lastly, the kinematic variability of symptomatic violinists was lower than that of asymptomatic violinists. Conclusion These found kinematic and kinetic differences could mean that violinists with overuse syndrome play the violin with less optimal motor control, resulting in larger loads on the joints and surrounding tissues. The lower variability of kinematics in violinists with pain may indicate an increase in stiffness. Overall, these findings suggest that biomechanical differences exist between violinists with and without overuse syndrome.