Background: Management of longitudinal forearm instability remains challenging. Chronic forearm stability may be overcome by reconstruction of the interosseous ligament (IOL). Despite the bands of the IOL being inseparable, studies of the IOL have focused on the central band (CB), but have neglected the proximal (PB) and distal (DB) bands. The purpose of this study was to characterize the bio-mechanical properties of the IOL. Materials and Methods: Twelve frozen specimens from individuals of both sexes were bio-mechanically analyzed using a custom-designed jig operated at constant angular speed to simulate forearm rotation. Strain was measured during dynamic forearm simulation using a motion tracking system. Results: The average strain of the CB, PB, and DB during forearm simulation were 0.08 ± 0.04, 0.83 ± 0.47, and 0.65 ± 0.23 mm (p < 0.001). The IOL was generally shortest during maximal pronation and increased as the forearm was rotated to a neutral position. The strain of the CB remain constant during forearm rotation and was the lowest at full pronation to 20° pronation position. Throughout forearm rotation, the strain of the CB remained constant, whereas the strain of the PB and DB fluctuated. Conclusions: The PB, CB, and DB of the forearm IOL have different bio-mechanical properties. CB maintained a constant rotational strain throughout forearm rotation. Strain on the CB was significantly lower than strains on the PB and DB. By contrast, strains on the PB and DB varied, suggesting that their roles differ from those of the CB. When CB reconstruction is needed, graft should be tensioned at 20° forearm pronation to gain optimum tension.