Influence of Image Acquisition and Biologic Variability on Measurement Reproducibility of Vastus Lateralis Muscle Architecture Using Ultrasound

Nacafucasaco, ETA, Da Silva, DDG, Bergamasco, JGA, Bittencourt, D, Chaves, TS, Scarpelli, MC, and Libardi, CA. Influence of image acquisition and biologic variability on measurement reproducibility of vastus lateralis muscle architecture using ultrasound. J Strength Cond Res 40(1): e108–e116, 2026—The objective of this study was to compare 2 reproducibility protocols for muscle architecture measurements of the vastus lateralis using ultrasonography (US): (a) a single image acquisition followed by 2 quantifications with a 72-hour interval (1X protocol) and (b) 2 separate image acquisitions followed by 2 quantifications, each 72 hours apart (2X protocol). A total of 54 subjects had both legs assessed by US, resulting in 104 experimental units. Muscle architecture parameters (cross-sectional area [CSA], muscle thickness [MT], fascicle length [FL], and pennation angle [PA]) were quantified, and typical error (TE), coefficient of variation (CV), minimum detectable change (MDC), and Bland–Altman plots were used for reproducibility analysis. Typical error and CV values were consistently lower for the 1X protocol (CSA: TE = 0.51 cm 2 , CV = 2.31%, MDC = 1.41 cm 2 ; MT: TE = 0.05 cm, CV = 2.61%, MDC = 0.14 cm; FL: TE = 0.21 cm, CV = 3.00%, MDC = 0.57 cm; PA: TE = 0.55°, CV = 3.68%, MDC = 1.52°) than for the 2X protocol (CSA: TE = 0.92 cm 2 , CV = 4.15%; MDC = 2.55 cm 2 ; MT: TE = 0.12 cm, CV = 6.22%, MDC = 0.33 cm; FL: TE = 0.48 cm, CV = 6.88%, MDC = 1.33 cm; PA: TE = 1.15°, CV = 7.82%, MDC = 3.18°). Paired t -tests and Wilcoxon tests indicated significant differences between protocols for FL ( p < 0.001) and PA ( p < 0.026), but not for CSA and MT. Levene's test showed greater variance in all parameters for the 2X protocol ( p < 0.001). These findings highlight the influence of reproducibility protocol on measurement variability and emphasize the need to account for evaluator-related and biologic sources of error in US-based muscle assessments.