Multiple studies indicated that the degree of muscle strain is the most relevant parameter in understanding the injury mechanism behind a hamstring strain injury. To monitor this parameter a new system is developed; the Smart Sensor Shorts. The system contains five Inertial Measurement Units (IMUs) integrated in a sports tights. The purpose of this study was to develop a methodology to estimate the muscle strain and muscle elongation velocity of the biceps femoris (BF), semimembranosus (SM) and the semitendinosus (ST) muscle in professional and recreational football athletes with the use of IMUs during different football specific movements and during different intensities. When comparing different movements and intensities, the greatest peak muscle strain was found in the BF and the lowest peak muscle strain was found in the SM during the majority of the movements. The biomechanical load was different for each hamstring muscle and was different for the running based movements and other football specific movements. The BF experienced the greatest peak muscle strain (12.12 ± 0.88%) during a maximal intensity kick in the supporting leg, while the greatest peak muscle elongation velocity was observed in the ST (3.97 ± 0.47 s-1) in the kicking leg during the maximal intensity kick. The greatest biomechanical loading was during a maximal intensity kick. Finally, it was observed that the moment of peak muscle strain was different from the time period of peak muscle elongation velocity for running based movements. It is concluded that IMUs together with the developed methodology could be used in the assessment of hamstring strain injuries in professional and recreational football by analyzing and monitoring muscle strain and muscle elongation velocity.