Despite the increase in research of hamstring stiffness through the use of ultrasound-based shear wave elastography, the active stiffness of biceps femoris long head (BFlh) and semitendinosus (ST) muscles under fatigue conditions at various contraction intensities has not been sufficiently explored. This study aimed to compare the effects of knee flexor’s isometric contraction until exhaustion performed at 20% vs. 40% of maximal voluntary isometric contraction (MVIC), on the active stiffness responses of BFlh and ST. Eighteen recreationally active males performed two experimental sessions. The knee flexors’ MVIC was assessed before the fatiguing task, which involved a submaximal isometric contraction until failure at 20% or 40% of MVIC. Active muscle stiffness of the BFlh and ST was assessed using shear wave elastography. BFlh active stiffness remained relatively unaltered at 20% of MVIC, while ST active stiffness decreased from ≅ 91% contraction time (55.79 to 44.52 kPa; p < 0.001). No intramuscular stiffness changes were noted in BFlh (36.02 to 41.36 kPa; p > 0.05) or ST (63.62 to 53.54 kPa; p > 0.05) at 40% of MVIC session. Intermuscular active stiffness at 20% of MVIC differed until 64% contraction time (p < 0.05) whereas, at 40% of MVIC, differences were observed until 33% contraction time (p < 0.05). BFlh/ST ratios were not different between intensities (20%=0.75 ± 0.24 ratio vs. 40%=0.72 ± 0.32 ratio; p > 0.05), but a steeper increase in BFlh/ST ratio was found for 20% (0.004 ± 0.003 ratio/%) compared to 40% (0.001 ± 0.003 ratio/%) of MVIC (p = 0.003). These results suggest that contraction duration could play a major role in inducing changes in hamstrings’ mechanical properties during fatigue tasks compared to contraction intensity.