Background:All-inside anterior cruciate ligament reconstruction (ACLR) is an emerging technique used to treat ACL injuries. The all-inside technique uses a 4-stranded graft made from a single tendon that is looped on itself. The 4 strands of the graft must be secured to each other to become a closed-loop structure. Various suture configurations exist to secure the graft to adjustable loop devices, and there is a lack of data to support one technique over another. In addition to the primary sutures used to fasten the graft together, accessory sutures can be tied over the button as secondary fixation.Purpose:To evaluate biomechanical properties of 4-stranded grafts prepared in 5 different configurations.Study Design:Controlled laboratory study.Methods:Porcine flexor tendons (N = 25) were prepared in 5 different configurations (n = 5 tendons per group): simple-interrupted sutures (unsecured fixation), side-to-side fixation with and without secondary fixation, and end-to-end fixation with and without secondary fixation. The grafts were put through the same mechanical testing protocol (cyclic loading, pull to failure) to measure average load at graft failure, average displacement at failure, average stiffness, and average elongation rate. Differences between graft preparation techniques were investigated using 1-way analyses of variance (ANOVAs) with post hoc t tests (P < .05).Results:Significant 1-way ANOVAs for each biomechanical property were found. Unsecured fixation was the weakest graft preparation with the lowest stiffness (167 ± 12 N/mm), lowest ultimate failure load (637 ± 99 N), and highest elongation rate (0.0033 ± 0.0007 mm/s). End-to-end fixation without secondary fixation showed the highest ultimate failure load (846 ± 26 N), highest stiffness (212 ± 10 N/mm), and lowest rate of elongation (0.0025 ± 0.0001 mm/s). End-to-end fixation, both with and without secondary fixation, as well as side-to-side fixation with secondary fixation showed significantly higher ultimate failure loads than grafts with unsecured fixation. End-to-end fixation performed better than side-to-side fixation; however, for most variables, the difference was not statistically significant. Secondary fixation did not provide significant improvement.Conclusion:The all-inside ACL graft with simple-interrupted sutures is biomechanically inferior to a graft that has its free ends secured to the adjustable tibial loop. Adding secondary fixation to the tibial button does not significantly change the biomechanical properties. Further clinical studies are required to determine whether these findings translate into differences in clinical outcome.Clinical Relevance:All-inside ACLR is gaining popularity in hamstring ACL reconstructive techniques. These results provide surgeons with guidance on the best graft preparation method when using a single quadrupled hamstring tendon graft.