Eccentric quasi-isometric (EQI) resistance training is emerging as a promising option in sports medicine and rehabilitation. Despite prior research on EQI contractions in quadriceps and biceps brachii, their use in hamstring injury contexts is underexplored. Therefore, our study examines and contrasts the biomechanics and fatigue effects of EQI training on knee extensors and flexors. Following familiarization, 16 healthy, active participants (9 men, 7 women; 23.5 ± 2.6years, 72.1 ± 12.8kg, 173.4 ± 10.7cm) performed, in random order, four EQI contractions for knee extensions and flexions, respectively. EQI contractions were isotonically loaded to 70% of concentric (60°·s-1) maximal voluntary contraction. Rest between repetitions was set at three minutes, while four minutes separated each muscle group. Peak torque, mean torque, and optimal angle were evaluated pre- and post-bouts. Inter-repetition contraction time and angular velocity were also assessed. Average torque was 160.9 ± 44.2 and 71.5 ± 23.2 Nm for the extensors and flexors. Peak and mean torque significantly decreased for both extensors (p < 0.001, d = 0.70-0.71) and flexors (p ≤ 0.022, d = 0.36)after EQI contractions, respectively. However, the optimal angle increased for extensors (p < 0.001, d = 1.00) but not flexors (p = 0.811, d = 0.06). During EQI contractions, knee flexors exhibited greater intra-repetition velocity than extensors (p = 0.002; η2 = 0.50). Decreases in inter-repetition time and range of motion were more consistent for the extensors. Distinct responses exist when comparing EQI contractions of the knee extensors and flexors, particularly their effect onpeak torque angles. These findings suggest knee flexors may require lower relative intensities to align more closely with extensor EQI contractions.