REASONS FOR PERFORMING STUDY: Treadmill locomotion is frequently used for training of sport horses, for diagnostic purposes and for research. Identification of the possible biomechanical differences and similarities between the back movement during treadmill (T) and over ground (O) locomotion is essential for the correct interpretation of research results. OBJECTIVES: To compare the kinematics of the thoracolumbar vertebral column in treadmill and over ground locomotion in healthy horses. METHODS: Six sound Dutch Warmblood horses trotted on a T and O during 10 s at their own preferred velocity (mean +/- s.d. 3.6 +/- 0.3 m/s T and 3.6 +/- 0.1 m/s O), which was the same in both conditions. Kinematics of the vertebral column was captured by infrared cameras using reflective skin markers attached over the spinous processes of selected vertebrae and other locations. Flexion-extension and lateral bending range of motion (ROM), angular motion pattern (AMP) and intravertebral pattern symmetry (IVPS) of 5 vertebral angles (T6-T10-T13, T10-T13-T17, T13-T17-L1, T17-L1-L3 and L1-L3-15) were calculated. Neck angle, linear and temporal stride parameters and protraction-retraction angles of the limbs were also calculated. RESULTS: The vertical ROM (flexion-extension) was similar in both conditions, but the horizontal ROM (lateral bending) of the lumbar angles T17-L1-L3 and L1-L3-L5 was less during T locomotion (mean +/- s.d. difference of 1.8 +/- 0.6 and 1.7 +/- 0.9 degrees, respectively, P > 0.05). During O locomotion, the symmetry pattern of the lumbar vertebral angles was diminished from 0.9 to 0.7 (1 = 100% symmetry) indicating increased irregularity of the movement (P > 0.05). No differences were found in the basic linear and temporal stride parameters and neck angle. POTENTIAL RELEVANCE: Vertebral kinematics during treadmill locomotion is not identical to over ground locomotion, but the differences are minor. During treadmill locomotion lumbar motion is less, and caution should be therefore taken when interpreting lumbar kinematics.