The purpose of this study was to determine the effect, if any, that varying the distal testing position (tibial level) has on isometry data produced with a common anatomic proximal testing position at the native anterior cruciate ligament (ACL) origin. During ACL reconstruction in 25 knees, in vivo isometry measurements were recorded using two different isometry testing methods, which differed in the tibial level of the distal fixation testing point. Method 1 tested distally at a point 13 cm peripheral to the native ACL insertion on a vector in line with the tibial tunnel. Method 2 tested distally at a point central in the native ACL insertion at the level of the intercondylar floor. All tibial tunnels were standardized with similar sagittal tunnel-plateau angles and similar tunnel lengths. The proximal testing point was standardized at a point that was anatomically located at or near the central ACL origin 7 mm anterior to "over the top" in all knees. Using these methods, length changes between the proximal and distal testing points were recorded in each knee with each testing method, with the knee ranged from 70 degrees of flexion to full extension and from 70 degrees to 140 degrees of flexion. From these data, a total excursion from 0 degree to 140 degrees of flexion was calculated. A nonanatomic distal testing point (Method 1) produced a 6 mm +/- 1 mm total excursion, whereas anatomic testing points (Method 2) in the same knees produced a 1 mm +/- 1 mm total excursion. From these data, the authors conclude that the tibial level of the distal isometry testing point has a significant effect on the resultant isometry measurement such that anatomic testing points are most isometric. Isometers that produce data between nonanatomic testing points should not be used to position tunnels for ACL reconstruction and should not be used to assume the elongation forces an ACL substitute will see when fixed at different points. Conversely, the clinical relevance of this study is that both anatomic graft position and anatomic graft fixation position are important and, when achieved, should result in minimal graft elongation with early postoperative range of motion, leading to a more stable long-term result.