Mechanical alignment (MA) in total knee arthroplasty (TKA), although considered the gold standard, reportedly has up to 25% of patients expressing post-operative dissatisfaction. Biomechanical outcomes following kinematic alignment (KA) in TKA, developed to restore native joint alignment, remain unclear. Without a clear consensus for the optimal alignment strategy during TKA, the purpose of this study was to conduct a paired biomechanical comparison of MA and KA in TKA by experimentally quantifying joint laxity and medial collateral ligament (MCL) strain. 14 bilateral native fresh-frozen cadaveric lower limbs underwent medially-stabilised TKA (GMK Sphere, Medacta, Switzerland) using computed CT-based subject-specific guides, with KA and MA performed on left and right legs, respectively. Each specimen was subjected to sensor-controlled mediolateral laxity tests. A handheld force sensor (Mark-10, USA) was used to generate an abduction-adduction moment of 10Nm at the knee at fixed flexion angles (0°, 30°, 60°, 90°). A digital image correlation system was used to compute the strain on the superficial medial collateral ligament. A six-camera optical motion capture system (Vicon MX+, UK) was used to acquire kinematics using a pre-defined CT-based anatomical coordinate system. A linear mixed model and Tukey's posthoc test were performed to compare native, KA and MA conditions (p<0.05). Unlike MA, medial joint laxity in KA was similar to the native condition; however, no significant difference was found at any flexion angle (p>0.08). Likewise, KA was comparable with the native condition for lateral joint laxity, except at 30°, and no statistical difference was observed. Although joint laxity in MA seemed lower than the native condition, this difference was significant only for 30° flexion (p=0.01). Both KA and MA exhibited smaller MCL strain at 0° and 30°; however, all conditions were similar at 60° and 90°. Medial and lateral joint laxity seemed to have been restored better following KA than MA; however, KA did not outperform MA in MCL strain, especially after mid-flexion. Although this study provides only preliminary indications regarding the optimal alignment strategy to restore native kinematics following TKA, further research in postoperative joint biomechanics for load bearing conditions is warranted.