ABSTRACT Introduction: Recent studies have shown that the likelihood of semitendinosus-gracilis graft rupture is inversely correlated to its diameter. A graft can be prepared in a five-strand or four-strand fashion to increase its diameter. However, the biomechanical superiority of five-strand semitendinosus-gracilis grafts is still under debate. Objective: This study aimed to evaluate the biomechanical characteristics of matched four-strand and five-strand human semitendinosus-gracilis grafts. Methods: We evaluated semitendinosus-gracilis tendons harvested from ten fresh human male and female cadavers, aged 18-60 years. Four-strand or five-strand grafts were prepared with the tendons and fixed to wooden tunnels with interference screws. Each graft was submitted to axial traction at 20 mm/min until rupture; the tests were donor matched. Data were recorded in real time and included the analysis of the area, diameter, force, maximum deformation and stiffness of the grafts. Results: The diameter, area and tunnel size were significantly greater in the five-strand grafts than in the four-strand grafts. There were no significant differences in biomechanical properties. The area and diameter of the graft were positively correlated to stiffness, and inversely correlated to elasticity. There was no significant correlation between graft size and maximum force at failure, maximum deformation or maximum tension. Conclusion: Five-strand hamstring grafts have greater area, diameter and tunnel size than four-strand grafts. There were no significant differences in biomechanical properties. In this model using interference screw fixation, the increases in area and diameter were correlated with an increase in stiffness and a decrease in elasticity. Level of evidence V; biomechanical study.