Hamstring strain injuries are a prevalent burden in soccer. Low strength, muscle fatigue, and inter-limb differences in hamstring strength are associated with hamstring injury risk. Previous research shows increased hamstring injury incidence in soccer at the end of each half or the end of the match, respectively. This study aims at evaluating the aforementioned risk factors of hamstring injury over the course of a simulated soccer match. Ten active soccer players carried out the Loughborough Intermittent Shuttle Test, during which hamstring strength of both legs was assessed on seven occasions via the optimized 90:20 Isometric Posterior Chain Test. Hamstring strength of each limb and inter-limb differences in hamstring strength over the course of the Loughborough Intermittent Shuttle Test were parameters of interest. Repeated measures ANOVA were used to analyze the development of hamstring strength and limb-asymmetries in hamstring strength during the simulated soccer match. Compared to pre-match values, hamstring strength was significantly decreased after 15 and 30 minutes of simulated soccer match for the non-dominant and dominant leg, respectively. There were no further variations in hamstring strength within the simulated soccer match for either leg. We did neither measure significant recovery of hamstring strength to pre-match values at the beginning of the second half, as suspected by previous research, nor inter-limb differences, or a deterioration of limb asymmetries in hamstring strength during the simulated soccer match. Players who only participate for a short period in a soccer match may be exposed to the same risk of suffering hamstring injury like players who compete for a longer duration. Decreasing hamstring strength partly depicts the pattern of hamstring injury incidences during soccer matches. Additional factors may influence the increasing hamstring injury rate at the end of each half or the later stages of a match, respectively.