PurposeThe purpose of this study was to compare the biomechanics of 4-strand and 5-strand hamstring constructs for anterior cruciate ligament grafts.MethodsThirty-six human cadaveric hamstring grafts were tested in 3 different conditions: (1) graft femoral fixation complex, (2) graft femoral and tibial fixation (GFTF) complex using a human model, and (3) GFTF complex using a porcine model. Grafts were tested on a tensile testing machine. Four-stranded grafts served as the control group, and 5-stranded grafts served as the experimental group. Cyclic elongation, ultimate load to failure, stiffness, and diameter of the grafts were analyzed.ResultsAverage 4-strand graft diameter was 7.96 mm compared to 9.32 mm for the 5-strand graft (P = .00017). Average stiffness of grafts ≥8 mm was 105.04 N/mm compared to 85.05 N/mm for grafts <8 mm (P = .04988). There was a positive correlation between graft diameter and stiffness (13.4 N/mm per every 1 mm increase in diameter, r2 value of 13.1%, and F-significance of 0.02778). There were no significant differences in terms of ultimate load to failure, cyclic elongation, or stiffness between the experimental groups.ConclusionFive-strand hamstring grafts offer greater diameter and are biomechanically comparable to 4-strand equivalents at time 0. Grafts >8 mm offer significantly greater stiffness compared to grafts sized <8 mm. There is a weak positive correlation between graft diameter and stiffness.Clinical RelevanceA potential drawback to hamstring grafts is their variability in size. Five-strand hamstring grafts provide increased diameter in comparison to 4-strand equivalents and might be used when quadrupled graft diameter is <8 mm.