Introduction: The adolescent growth spurt is associated with rapid growth and hormonal changes, thought to contribute to the increased anterior cruciate ligament injury risk in girls. However, relatively little is known about these musculoskeletal and estrogen changes during the growth spurt in girls. Purpose: To investigate the longitudinal changes in estrogen as well as anterior knee laxity and lower limb strength and flexibility throughout the adolescent growth spurt in girls. Methods: Thirty-three healthy girls, age 10-13 yr, in Tanner stage II and 4-6 months from their peak height velocity were recruited. Participants were tested up to four times during the 12 months of their growth spurt, according to the timing of their maturity offset (test 1: maturity offset = -6 to -4 months; test 2: maturity offset = 0 months; test 3: maturity offset = +4 months; test 4: maturity offset = +8 months). During each testing session, anterior knee laxity, lower limb flexibility, and isokinetic strength as well as saliva measures of estradiol concentration were measured. Results: A significant (P = 0.002) effect of time on anterior knee laxity was found from the time of peak height velocity, although no changes in estradiol concentration were displayed over time (P = 0.811). Participants displayed a significant increase (P < 0.05) in isokinetic quadriceps strength over time, with no apparent increase in isokinetic hamstring strength. Conclusions: We speculate that increased quadriceps strength, combined with increased knee laxity and no accompanying hamstring strength development during the adolescent growth spurt in girls, might contribute to a decrease in their knee joint stability during landing tasks. These musculoskeletal changes could potentially increase anterior cruciate ligament injury risk at a time of rapid height and lower limb growth.