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The inter-relationship between equine thoracolumbar motion and muscle activation during normal locomotion and lameness is poorly understood.To compare thoracolumbar and pelvic kinematics and longissimus dorsi (longissimus) activity of trotting horses between baseline and induced forelimb (iFL) and hindlimb (iHL) lameness.Controlled experimental cross-over study.Three-dimensional kinematic data from the thoracolumbar vertebrae and pelvis, and bilateral surface electromyography (sEMG) data from longissimus at T14 and L1, were collected synchronously from clinically non-lame horses (n = 8) trotting overground during a baseline evaluation, and during iFL and iHL conditions (2-3/5 AAEP), induced on separate days using a lameness model (modified horseshoe). Motion asymmetry parameters, maximal thoracolumbar flexion/extension and lateral bending angles, and pelvis range of motion (ROM) were calculated from kinematic data. Normalised average rectified value (ARV) and muscle activation onset, offset and activity duration were calculated from sEMG signals. Mixed model analysis and statistical parametric mapping compared discrete and continuous variables between conditions (α=0.05).Asymmetry parameters reflected the degree of iFL and iHL. Maximal thoracolumbar flexion and pelvis pitch ROM increased significantly following iFL and iHL. During iHL, peak lateral bending increased towards the non-lame side (NLS) and decreased towards the lame side (LS). Longissimus ARV significantly increased bilaterally at T14 and L1 for iHL, but only at LS L1 for iFL. Longissimus activation was significantly delayed on the NLS and precipitated on the LS during iHL, but these clear phasic shifts were not observed in iFL.Findings should be confirmed in clinical cases.Distinctive, significant adaptations in thoracolumbar and pelvic motion and underlying longissimus activity occur during iFL and iHL and are detectable using combined motion capture and sEMG. For iFL, these adaptations occur primarily in a cranio-caudal direction, whereas for iHL, lateral bending and axial rotation are also involved. This article is protected by copyright. All rights reserved.