•Biceps femoris long head fascicle and sarcomere lengths increase after 3 weeks of Nordic hamstring exercise training.•Architectural adaptations occurred only in the distal portion of the muscle.•Increases in fascicle length in response to 3 weeks of exercise training were not the result of serial sarcomere addition. Eccentric exercise increases muscle fascicle lengths; however, the mechanisms behind this adaptation are still unknown. This study aimed to determine whether biceps femoris long head (BFlh) fascicle length increases in response to 3 weeks of eccentric exercise training are the result of an in-series addition of sarcomeres within the muscle fibers. Ten recreationally active participants (age = 27 ± 3 years; mass = 70 ± 14 kg; height = 174 ± 9 cm; mean ± SD) completed 3 weeks of Nordic hamstring exercise (NHE) training on a custom exercise device that was instrumented with load cells. We collected in vivo sarcomere and muscle fascicle images of the BFlh in 2 regions (central and distal) by using microendoscopy and 3 dimension ultrasonography. We then estimated sarcomere length, sarcomere number, and fascicle length before and after the training intervention. Eccentric knee flexion strength increased after the training (15%; p < 0.001; ηp2 = 0.75). Further, we found a significant increase in fascicle length (21%; p < 0.001; ηp2 = 0.81) and sarcomere length (17%; p < 0.001; ηp2 = 0.90) in the distal but not in the central portion of the muscle. The estimated number of sarcomeres in series did not change in either region. Fascicle length adaptations appear to be heterogeneous in the BFlh in response to 3 weeks of NHE training. An increase in sarcomere length, rather than the addition of sarcomeres in series, appears to underlie increases in fascicle length in the distal region of the BFlh. The mechanism driving regional increases in fascicle and sarcomere length remains unknown, but we speculate that it may be driven by regional changes in the passive tension of muscle or connective tissue adaptations. Display omitted