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Purpose: This study identified the co-activation of quadriceps and hamstring muscles during hamstring strengthening exercises in healthy adults. Methods: Twenty-one participants were required for the present study design to achieve 80% power, 0.8 effect size (η2), and an alpha level of 0.05. Thus, this study recruited 21 healthy adults. All participants performed Nordic exercises, bridge exercises, and one-leg deadlifts randomly. The activity of the rectus femoris, vastus medialis (VM), vastus lateralis (VL), biceps femoris (BF), and semitendinosus (SM) were measured. In addition, the ratios of VM/VL and hamstring/quadriceps (HQ) were measured during the three hamstring strengthening exercises using electromyography. One-way ANOVA was used to compare the co-activation of quadriceps and hamstring muscles in the three exercises. Results: The activity of VM and VL during the performance of one-leg deadlifts was significantly higher than the other two exercises. The BF had significantly higher activity during the Nordic exercises compared to the other two exercises. In addition, the SM activation was significantly greater during Nordic exercises than one-leg deadlifts. Additionally, there was significant difference in HQ ratio among hamstring strengthening exercises. In specific, the one-leg deadlifts yielded a significantly lower HQ ratio. Conclusion: This study revealed that one-leg deadlifts are effective in rehabilitation for anterior cruciate ligament injury. In addition, Nordic exercises can be recommended to facilitate hamstring muscle activation.