Prior studies have reported a negative effect on both clinical outcomes and patient-reported outcome measures (PROMS) following joint line elevation (JLE) in cruciate-retaining (CR) total knee arthroplasty (TKA) and posterior stabilized (PS) TKA designs. This experimental study was aimed to quantify the effect of JLE on in vivo knee kinematics in patients with bicruciate retaining (BCR) TKA during strenuous activities. Thirty unilateral BCR TKA patients were evaluated during single-leg deep lunge and sit-to-stand using a validated combined computer tomography and dual fluoroscopic imaging system. Correlation analysis was performed to quantify any correlations between JLE and in vivo kinematics, as well as PROMS. There was a significant negative correlation between JLE and maximum flexion angle during single-leg deep lunge (ρ = −0.34, p = 0.02), maximum varus joint angles during single-leg deep lunge (ρ = −0.37, p = 0.04), and sit-to-stand (ρ = −0.29, p = 0.05). There was a significant negative correlation between JLE and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score (ρ = −0.39, p = 0.01) and knee disability and osteoarthritis outcome score physical function (KOOS-PS; ρ = −0.33, p = 0.03). The JLE that yields a significant loss in PROMS and maximum flexion angles were 2.6 and 2.3 mm, respectively. There was a linear negative correlation of JLE with both in vivo knee kinematics and PROMS, with changes in JLE of greater than 2.6 and 2.3 mm, leading to a clinically significant loss in PROMS and maximum flexion angles, respectively, suggesting an increased need to improve surgical precision to optimize patient outcomes following BCR TKA.