PURPOSE To compare biceps femoris long-head (BFlh) fascicle lengths (Lfs) obtained with different ultrasound-based approaches: 1) single ultrasound images and linear Lf extrapolation; 2) single ultrasound images and one of two different trigonometric equations (termed equations A and B); and 3) extended field of view (EFOV) ultrasound images. METHODS Thirty-seven elite alpine skiers (21.7±2.8 yrs) without a previous history of hamstring strain injury were tested. Single ultrasound images were collected with a 5 cm linear transducer from BFlh at 50% femur length and were compared with whole muscle scans acquired by EFOV ultrasound. RESULTS The intra-session reliability (ICC3,k = intraclass correlation coefficient) of Lf measurements was very high for both single ultrasound images (i.e., Lf estimated by linear extrapolation; ICC3,k = 0.96-0.99, SEM = 0.18 cm) and EFOV scans (ICC3,k = 0.91-0.98, SEM = 0.19 cm). Although extrapolation methods showed cases of overestimation and underestimation of Lf when compared with EFOV scans, mean Lf measured from EFOV scans (8.07±1.36 cm) was significantly shorter than Lf estimated by trigonometric equations A (9.98±2.12 cm, P<0.01) and B (8.57±1.59 cm, P=0.03), but not significantly different from Lf estimated with manual linear extrapolation (MLE) (8.40±1.68 cm, p=0.13). Bland-Altman analyses revealed mean differences in Lfs obtained from EFOV scans and those estimated from equation A, equation B and MLE of 1.91±2.1 cm, 0.50±1.0 cm and 0.33±1.0 cm, respectively. CONCLUSIONS The typical extrapolation methods used for estimating Lf from single ultrasound images are reliable within the same session, but not accurate for estimating BFlh Lf at rest with a 5-cm FOV. We recommend that EFOV scans are implemented to accurately determine intervention-related Lf changes in BFlh.