The present study aimed to compare muscle architecture and electromyographic activity during isotonic (IT) and isokinetic (IK) knee extensors eccentric contractions. Seventeen subjects were assigned in test and reproducibility groups. During test session, subjects performed two IT and two IK sets of eccentric contractions of knee extensor muscles. Torque, angular velocity, VL architecture and EMG activity of agonist (vastus lateralis, VL; vastus medialis; rectus femoris) and antagonist (semitendinosus; biceps femoris, BF) muscles were simultaneously recorded and averaged on a 5° window. Torque-angle and angular velocity-angle relationships exhibited differences in mechanical load between the IT and IK modes. Changes in muscle architecture were similar in both modes, since VL fascicles length increased and fascicle angle decreased, resulting in a decrease in muscle thickness during eccentric contraction. Agonist activity and BF co-activity levels were higher in IT mode than in IK mode at short muscle lengths, whereas agonist activity was higher in IK mode than in IT mode at long muscle lengths. Differences in mechanical load between both modes induced specific neuromuscular responses in terms of agonist activity and antagonist co-activity. These results suggest that specific neural adaptations may occur after IT or IK eccentric training. This hypothesis needs to be tested in order to gain new insights concerning the most effective eccentric protocols based on whether the objective is sportive or clinical.