The present study examined the reliability and validity of in vivo vastus lateralis (VL) fascicle length (L(f)) assessment by extended field-of-view ultrasonography (EFOV US). Intraexperimenter and intersession reliability of EFOV US were tested. Further, L(f) measured from EFOV US images were compared to L(f) measured from static US images (6-cm FOV) where out-of-field fascicle portions were trigonometrically estimated (linear extrapolation). Finally, spatial accuracy of the EFOV technique was assessed by comparing L(f) measured on swine VL by EFOV US to actual measurements from digital photographs. The difference between repeated VL L(f) measurements by the same experimenter was 2.1 ± 1.7% with an intraclass correlation (ICC) of 0.99 [95% confidence interval (CI) = 0.95-1.00]. In terms of intersession reliability, no difference (P = 0.48) was observed between L(f) measured on two different occasions, with ICC = 0.95 (CI = 0.80-0.99). The average absolute difference between L(f) measured by EFOV US and using linear extrapolation was 12.6 ± 8.1% [ICC = 0.76 (CI = -0.20-0.94)]; EFOV L(f) was always longer than extrapolated L(f). The relative error of measurement between L(f) measured by EFOV US and by dissective assessment (digital photographs) in isolated swine VL was 0.84% ± 2.6% with an ICC of 0.99 (CI = 0.94-1.00). These results show that EFOV US is a reliable and valid method for the measurement of long muscle fascicle in vivo. Thus EFOV US analysis was proven more accurate for the assessment of skeletal muscle fascicle length than conventional extrapolation methods.