Assessment of Lower Limb Muscle Volume Using 3D Ultrasonography: Validity and Reliability Compared to MRI
Authors: Ritsche Paul , Ledergerber Romina , Pansini Michele , Santini Francesco , Faude Oliver 
Published: January 2025

Introduction Muscle volume is a crucial indicator of muscle strength and neuromuscular health, traditionally assessed using Magnetic Resonance Imaging (MRI). While MRI provides high accuracy, it is costly and time-consuming. Three-dimensional ultrasonography (3DUS) offers a more accessible alternative but requires rigorous validation due to setup dependent accuracy. This study evaluates the validity and reliability of a custom 3DUS setup for measuring lower limb muscle volumes, compares it to MRI, and documents the methodology for broader research adoption.
Methods We recruited 10 healthy participants (5 males, 5 females; 18–40 years). The protocol included two 3DUS sessions and one MRI session spaced one week apart. The 3DUS scans were acquired by one experienced operator, while two operators analyzed the captured volumes from the second scanning timepoint. Target muscles—biceps femoris, gastrocnemius medialis and lateralis, tibialis anterior, and vastus lateralis—were scanned using a linear probe (12L3, Siemens, Erlangen, Germany) integrated with a motion capture system (Optitrack Flex 3, NaturalPoint, Corvallis, OR, USA). Data synchronization and reconstruction were performed using 3D Slicer. MRI scans were acquired using a 3T whole-body scanner (MAGNETOM Prisma, Siemens, Erlangen, Germany), and muscle volumes were segmented using Dafne and 3D-Slicer software. Validation was enhanced with phantom models (100–600 mL) scanned via both modalities. Reliability metrics included intra-class correlation (ICC), coefficients of variation (CV%), standard error of measurement (SEM), minimal detectable change (MDC) and standardized mean bias.
Results Test-retest reliability of 3DUS was high for all muscles, with ICCs ranging from 0.97 to 0.99 (95% confidence intervals between 0.86–0.99) and CV% values between 2.0 and4.6% (95% CI between 1.3–6.7%). MDC values were below 5 mL for all muscles, indicating sensitivity to detect small volume changes. The biceps femoris exhibited the highest test-retest reliability, while the gastrocnemius medialis showed lower but acceptable agreement. Inter-rater reliability was similarly high, with ICCs exceeding 0.97 (95% CIs between 0.51–0.99) for all assessed muscles and minimal variability between operators (CV%: 2.1–3.2%, 95% CIs between 1.1–4.7%). Tibialis anterior yielded the highest inter-rater reliability results, vastus lateralis the lowest. Compared to MRI, 3DUS underestimated muscle volumes, with mean biases ranging from -15.4% to -44.6%. Agreement was highest for the tibialis anterior and lowest for the gastrocnemii. Standardized mean biases were classified as moderate to very large, ranging from -0.92 to -2.64. In contrast, phantom scans confirmed high accuracy for both 3DUS and MRI, suggesting measurement errors in vivo were likely due to probe pressure and sweep inconsistencies.
Discussion/Conclusion Our 3DUS system demonstrated excellent reliability for assessing muscle volumes between sessions and raters, but showed limited comparability to MRI, systematically underestimating volumes. The magnitude of underestimation appeared to be influenced by muscle architecture and location. Calibration refinements, including reduced probe pressure and optimized sweep protocols, are underway to improve accuracy. Nonetheless, 3DUS is a promising, cost-effective tool for longitudinal muscle assessments, particularly for detecting intervention-induced changes. Our openly accessible documentation of the methodology supports reproducibility and further innovation in the field.