Hamstring injuries are a major problem in sports involving sprinting, such as soccer, rugby, and track and field, and lead to sports stoppages and psychological, social, and financial repercussions. For several years now, these injuries have been stagnating or even increasing. Preventing these injuries is therefore a fundamental issue for at-risk athletes. The aim of this study was to compare the electromyographic(EMG) activity of the hamstrings in athletes during sprinting, Nordic hamstring exercise (NHE), and high-speed concentric exercise on an isokinetic dynamometer. A pilot study was conducted on a population of 15 sprint-exposed field athletes (22.54 ± 3.71 years, Tegner score ≥ 6) with no history of hamstring injury in the last six months. The protocol included a warm-up, followed by three repetitions of the NHE, two sets of 10 repetitions on the isokinetic dynamometer at 300°/sec, and a maximal sprint. Exercises were randomized for each subject, and muscle activity was assessed using wireless EMG sensors during each test. EMG data were normalized to the measured maximum voluntary isometric contraction (MVIC), and test results were statistically analyzed to determine which exercise resulted in maximal hamstring activation. Comparison of hamstring muscle activity between exercises showed a significant difference for most of our results (p-value < 0.05). The results show significantly higher mean hamstring activity during sprinting (0.4800 ± 0.19 μV) compared with strengthening exercises. The NHE (0.3201 ± 0.09 μV) was the second most active exercise. In the last place was the high-speed concentric exercise on the isokinetic machine, which produced less activation than the other exercises (0.2487 ± 0.07 μV). Sports involving sprinting are at risk of a hamstring injury but it appears that its use in rehabilitation and prevention of hamstring injury is relevant, as it would allow high-intensity muscle activation to prepare the hamstring for this type of loading. However, it is also fundamental to integrate strengthening exercises such as NHE in combination with sprinting in our rehabilitation. Finally, the use of the isokinetic dynamometer does not constitute a first-line choice for hamstring injury management.