PurposeA large percentage of anterior cruciate ligament (ACL) surgical reconstructions experience sub-optimal outcomes within 2 years. A potential factor contributing to poor outcomes is an incomplete understanding of micro-level, regional ACL biomechanics. This research aimed to demonstrate a minimally invasive method that uses micro-CT imaging to quantify regional ACL strains under clinically relevant joint loading.MethodsA pattern of 0.8 mm diameter zirconium dioxide beads were arthroscopically inserted into four regions of the ACL of four cadaveric knee specimens (mean [SD] age = 59 [9] years). A custom micro-CT compatible joint motion simulator then applied clinically relevant joint loading conditions, while an image was acquired at each condition. From the resulting images, strains within each region were calculated using the centroid coordinates of each tissue-embedded bead. Strain repeatability was assessed using the mean intra-specimen standard deviation across repeated load applications. A one-way repeated measures ANOVA (α = 0.05) was used to determine regional strain variations.ResultsThe mean intra-specimen standard deviation across repeated load application was ±0.003 strain for all specimens. No statistically significant differences were found between tissue regions, although medium and large effect sizes (0.095–0.450) suggest that these differences may be clinically relevant.ConclusionsThe method presented here demonstrates a minimally invasive measurement of regional ACL strain under clinically relevant joint loads using micro-CT imaging. The strain measurements demonstrated excellent reliability across the five repeated load applications and suggest a non-homogenous distribution of strain through the ACL.