Despite the advancement of diagnostic surgical techniques in anterior cruciate ligament (ACL) reconstruction and rehabilitation protocols following ACL injury, only half of the athletes return to sports at a competitive level. A major concern is neuromechanical dysfunction, which occurs with injuries persisting in operated and non-operated legs following ACL rehabilitation. One of the criteria for a safe return to sports participation is based on the maximal voluntary isometric contraction (MVIC) performed unilaterally and a comparison between the 'healthy knee' and the 'operated knee'. The present study aimed to investigate MVIC in athletes following ACL rehabilitation during open kinetic chain exercise performed unilaterally and bilateral exercises. Twenty subjects participated in the present investigation: 10 male athletes of regional-national level (skiers, rugby, soccer, and volleyball players) who were previously operated on one knee and received a complete rehabilitation protocol (for 6-9 months) were included in the ACL group (age: 23.4 ± 2.11 years; stature: 182.0 ± 9.9 cm; body mass: 78.6 ± 9.9 kg; body mass index: 23.7 ± 1.9 kg/m2), and 10 healthy male athletes formed the control group (CG: age: 24.0 ± 3.4 years; stature: 180.3 ± 10.7 cm; body mass: 74.9 ± 13.5 kg; body mass index: 22.8 ± 2.7 kg/m2). MVICs synchronised with electromyographic (EMG) activity (recorded on the vastus lateralis, vastus medialis, and biceps femoris muscles) were performed during unilateral and bilateral exertions. The rate of force development (RFD) and co-activation index (CI) were also calculated. The differences in the MVIC and RFD between the two legs within each group were not significant (p > 0.05). Vastus lateralis EMG activity during MVIC and biceps femoris EMG activity during RFD were significantly higher in the operated leg than those in the non-operated leg when exertion was performed bilaterally (p < 0.05). The CI was higher in the operated leg than that in the non-operated leg when exertion was performed bilaterally (p < 0.05). Vice versa, vastus medialis EMG activity during RFD was significantly higher in the right leg than that in the left leg when exertion was performed bilaterally (p < 0.05) in the CG. MVICs performed bilaterally represent a reliability modality for highlighting neuromechanical asymmetries. This bilateral exercise should be included in the criteria for a safe return to sports following ACL reconstruction.