Purpose: Approximately one-third of individuals with an anterior cruciate ligament reconstruction (ACLR) develop post-traumatic knee osteoarthritis (PTOA) within the first decade following ACLR. Currently there is a need to develop methods of early detection of PTOA, as articular cartilage damage is irreversible. Diagnostic brightness-mode (B-mode) ultrasound has been used to evaluate femoral cartilage deformation, evidenced by a decrease in cross-sectional area (CSA), following 30 minutes of treadmill walking in healthy uninjured individuals. Additionally, femoral cartilage CSA is greater in the ACLR limb compared to the uninjured limb in a resting state, which is similar to results from magnetic resonance imaging studies hypothesized to demonstrate cartilage swelling following joint injury. It remains unknown if the magnitude of femoral cartilage deformation following a standardized walking protocol is related to clinically relevant symptoms (CRS) in those with an ACLR. Persistent CRS, measured with the Knee Osteoarthritis and Injury Outcome Score (KOOS), have been reported in 39% of individuals six years following ACLR. The purpose of the current study was to determine the magnitude of femoral cartilage deformation following a standardized walking protocol which best-identified individuals with CRS. We hypothesized that individuals with this magnitude of femoral cartilage deformation would also be more likely to demonstrate CRS. Methods: Thirty-two individuals (56% female, 22±4 years old, 23.8±2.8 kg/m2body mass index, 50±37 months since ACLR) with a history of primary unilateral ACLR were recruited for a cross-sectional descriptive laboratory experiment. Femoral cartilage CSA was measured with B-mode ultrasound immediately prior to and following 3,000 steps of walking on a treadmill at a self-selected comfortable speed. Prior to the pre-walking ultrasound images, individuals rested in the long-sit position for 45 minutes to allow the cartilage rebound for a baseline CSA assessment. Individuals moved their knee into 140° of flexion to capture the anterior portion of the femur on the ultrasound during imaging. Femoral cartilage was segmented using ImageJ software, and cartilage deformation on the involved limb was calculated as a percent change (%Δ)from baseline, with negative %Δ indicating deformation following walking. All individuals were classified as symptomatic or asymptomatic based on previously defined cutoff values for the KOOS. A symptomatic individual was defined as scoring ≤ 87.5 on the quality of life portion of the KOOS as well as scoring below the cutoff of at least two other subscales of the KOOS questionnaire (KOOS Symptoms ≤ 85.7; KOOS Pain ≤ 86.1; KOOS ADL ≤ 86.8, KOOS Sports ≤ 85.0). Fourteen of the 32 individuals were classified as symptomatic. Area under the curve (AUC) values were calculated using receiver operator characteristic curve analysis to determine the capacity of femoral cartilage deformation to identify symptomatic individuals. If a significant AUC was determined (i.e. AUC 95% confidence intervals [CI] did not cross 0.5), we identified the cutoff value maximizing sensitivity and specificity. Lastly, an odds ratio was calculated to demonstrate the association between the cartilage deformation cutoff score and symptoms. Results: Femoral cartilage deformation -0.15±4.47%Δ, range: -8.18 - 13.87%Δ) displayed fair accuracy (AUC = 0.70; 95% CI, 0.51 - 0.89) for identifying symptomatic individuals. A femoral cartilage deformation cutoff of -0.29% was found to maximize sensitivity (0.79) and specificity (0.67). Individuals whose femoral cartilage did not deform by at least -0.29%Δ (meaning cartilage CSA stayed the same or increased during walking) displayed 7.33 (1.47 - 36.66) times higher odds of being symptomatic. Conclusions: Contrary to our hypothesis, individuals who did not demonstrate deformation of at least -0.29%Δ in the involved limb during 3,000 steps were more likely to report CRS. Sixteen individuals (50%) did not demonstrate deformation and experienced an increase in CSA during walking. Overall, while the mean is small and the standard deviation is large, this reflects half of participants increased CSA while the other half decreased CSA during walking. Individuals who demonstrated an increase in femoral cartilage CSA following walking were more likely to be symptomatic. Preliminary data demonstrates femoral cartilage in uninjured controls deforms (-1.33±1.65%) following 3,000 steps, which may indicate that femoral cartilage deformation is a healthy response to walking. The mechanism leading to an increase in CSA following 3,000 steps in these ACLR individuals remains unknown. It is possible increased CSA following walking is due to altered cartilage composition or aberrant biomechanics, which may promote cartilage swelling or altered fluid shifts within the tissue during movement.