Current calcaneal quantitative ultrasound systems assess different regions of interest (ROI), under different levels of lower limb loading, yield different parameter values, and are likely prone to different levels of error. This study evaluated the repeatability of measures of frequency-dependent attenuation (FDA, 0.3-0.8 MHz) at three calcaneal ROI, Brooke-Wavell (BW), Jaworski (JA), and foot gauge (FG), under four loading conditions (non-weightbearing, semi-weightbearing, bipedal stance, and unipedal stance). FDA in the calcaneus was assessed in 20 healthy participants (mean (± SD) age, 41.7 ± 19.6 years
height, 1.70 ± 0.16 m
and weight, 70.1 ± 23.0 kg) using a custom-built transmission-mode ultrasound system. Reliability was evaluated using the standard error of measurement (SEM) and limits of agreement (LA) and tolerance (95%TL). Differences in mean FDA values between ROI, loading, and measurement occasions were assessed using a repeated measures ANOVA (α = .05). Mean FDA values ranged between 58.0 ± 32.0 and 77.2 ± 27.6 dB/MHz across all conditions. Repeatability of FDA was dependent on the ROI examined and tended to improve with weightbearing. The narrowest limits for 95%TL ranged between ± 15.1 dB/MHz (JA SWB) and ± 62.7 dB/MHz (BW NWB) across sites. The SEM was approximately 10 dB/MHz for both FG and JA during non-weightbearing and was reduced to around 5 dB/MHz with full weightbearing. This study demonstrates that, although measures of ultrasound FDA are dependent on the ROI, lower limb loading may be a useful method to improve the repeatability of FDA measurements.