Purpose: A complex association exists between aberrant gait biomechanics and the development of posttraumatic osteoarthritis (PTOA) following anterior cruciate ligament reconstruction (ACLR). Therefore, a comprehensive understanding of the association between gait biomechanics and tibiofemoral articular cartilage composition is needed to develop the most appropriate biomechanical interventions to prevent PTOA post-ACLR. Recent waveform analyses demonstrate that differences in gait biomechanics exist between ACLR patients and uninjured, matched-controls at multiple portions of the stance phase in the first 12 months post-ACLR. Specifically, ACLR individuals demonstrate lesser vertical ground reaction force (vGRF) in early (i.e., 1-30% of stance) and late stance (i.e., 70-100% of stance) and greater vGRF during midstance (i.e., 33-64% of stance) compared to uninjured counterparts. Unfortunately, the majority of previous literature has focused on the influence of peak loading in early stance on PTOA development, leading to a dearth of knowledge regarding the link between mid and late stance loading and PTOA development. Therefore, the purpose of this exploratory study was to determine the associations between vGRF, a biomechanical indicator of lower limb loading, throughout the entirety of stance phase with in vivo estimates of tibiofemoral articular cartilage proteoglycan density using T1rho magnetic resonance imaging (MRI) relaxation times in individuals who were 12 months post-ACLR. Methods: Twenty-three participants (48% female, 22.09±4.09 years old, 24.18±3.30 kg/m2 body mass index) with unilateral ACLR participated in this cross-sectional study. vGRF was collected barefoot during an overground walking task at self-selected walking speed across a 6m walkway. vGRF data were time normalized to 101 unique points of stance phase between heel strike and toe off and normalized to body weight (BW). MRI was collected on either a Siemens Magnetom TIM Trio 3T or a Siemens Magnetom Prisma 3T scanner using a T1rho prepared three-dimensional Fast Low Angle Shot (FLASH) sequence with a spin-lock power at 500Hz at five different spin-lock durations (40, 30, 20, 10, 0 ms). Voxel-by-voxel T1rho relaxation maps were constructed from a five-image sequence using an in-house program. Anterior, central and posterior regions of interest (ROI) were manually segmented from the weightbearing the articular cartilage of the medial (MFC) and lateral femoral (LFC) and tibial (MTC and LTC) condyles. ROI were determined by the location of the meniscus in the sagittal plane and included: 1) the articular cartilage communicating with the anterior horn of the meniscus (anterior MFC/LFC and MTC/LTC), 2) the central portion of the articular cartilage between the anterior and posterior horns of the meniscus (central MFC/LFC and MTC/LTC), and 3) the articular cartilage communicating with the posterior horn of the meniscus (posterior MFC/LFC and MTC/LTC). Primary analysis utilized a global weightbearing score for T1rho relaxation times averaged across the three ROI (anterior, central and posterior) for each condyle. Greater T1rho MRI relaxation times are interpreted as lesser cartilage proteoglycan density. In an exploratory manner, we conducted our primary analyses with separate bivariate, Pearson Product Moment correlation coefficients (r) between T1rho relaxation times for each global region and vGRF at each 1% increment of stance phase (1-101%). Additionally, we constructed corresponding 95% confidence intervals (CI) for all Pearson Product Moment correlation coefficients at each 1% of stance phase using a Fisher’s transformation. We recognized associations as weak (r=0.0 to 0.3), moderate (r=0.3 to 0.5) or strong (r>0.5). We focused the discussion of our primary analyses on associations demonstrating Pearson Product Moment correlation coefficients with 95% CI that did not include zero. For easy visualization purposes, we presented the magnitude of Pearson Product Moment correlation coefficients and corresponding 95% CI (y-axis) at each percentage of stance (x-axis) for each of the femoral and tibial condyles (Fig. 1). If 95% CI were found not to cross zero for a correlation coefficient in primary analyses of global weightbearing regions, we conducted secondary Pearson Product Moment correlation analyses in the same manner as our primary analyses to further determine whether T1rho MRI relaxation times in a specific weightbearing ROI (anterior, central, posterior) were associated with vGRF. Results: Greater vGRF during the midstance of gait (46-56% of stance phase) was associated with greater MFC T1ρ MRI relaxation times (r ranging between 0.43 and 0.46) with corresponding 95% CI that did not include zero (Fig. 1a). Secondary analyses demonstrated that only weak associations (r ranging between -0.29 and 0.29) were observed between vGRF and anterior MFC. Greater vGRF was associated with greater T1rho MRI relaxation times in the central MFC (r ranging between r=0.43 and 0.50) during midstance (45-55% of stance), while lesser vGRF was associated with greater T1rho MRI relaxation times in the central MFC (r ranging between -0.43 and -0.45) during late stance (74-78% of stance). Greater vGRF was associated with greater T1rho MRI relaxation times in the posterior MFC (r ranging between 0.43 and 0.54) during midstance (36-53% of stance). Weak to moderate associations were found in the LFC (Fig. 1b), MTC (Fig. 1c), and LTC (Fig. 1d) regions; however, all associated 95% CIs included zero. Conclusions: Greater lower extremity loading during midstance demonstrated the strongest associations with articular cartilage T1rho MRI relaxation times in the MFC. Our secondary analyses further demonstrate that the association between greater vGRF during midstance and greater T1rho MRI relaxation times was strongest in the central and posterior MFC ROIs. Adequate loading and unloading of articular cartilage are critical to maintain optimal articular cartilage health. Our data suggest that inadequate unloading of the articular cartilage during midstance is linked to lesser proteoglycan density in the MFC post-ACLR. Previous data demonstrate that individuals with ACLR walk with a less dynamic vGRF waveform compared to uninjured, matched-controls, characterized by lower vGRF peaks in early and late stance and greater vGRF in midstance. Therefore, it is possible that dynamically unloading the lower extremity during midstance may result in optimal MFC articular cartilage health and promote greater proteoglycan density within the tissue. Overall, our study suggests the relation between gait biomechanics and tibiofemoral articular cartilage composition varies across stance phase and future work should evaluate the association between midstance kinetics and PTOA development.