Flexibility is considered as an important factor of physical fitness among all athletes, which can be improved via static or dynamic type, muscle elongation. Although the ideal duration of static muscle elongation has been extensively researched, no one has examined the effect of dynamic muscle elongation and more precisely the proper duration of mat Pilates exercises required for flexibility improvement. The aim of the present study was to assess the acute effects of a low number of repetitions of mat Pilates exercises, performed in a single training session, on the joint flexibility of female amateur soccer players. The sample consisted of 16 volunteers, female amateur soccer players, with a mean age of 26.2±3.8 years old, height of 169.0±3.0 cm, body mass of 67.2±5.8 kg and 14.2±2.8 mean training years, all familiar with Pilates. Participants performed two distinct mat Pilates protocols on non-consecutive sessions, in random order. The first protocol consisted of initial flexibility measurements, selected mat Pilates exercises repeated 5 times (1x5)in a full range of motion (ROM) and final flexibility measurements. The second protocol was similar to the first, expect for the number of repetitions which were double (2x5) and the break intervals between sets lasted for 5 sec. ROM was measured passively during hip flexion, extension and abduction, knee flexion and ankle dorsiflexion on the right side of the body, with a flexometer. A 2x2 Analysis of Variance with repeated measures was applied to assess differences in the flexibility pre- and post-measurements, for both Pilates protocols. The results revealed that the selected mat Pilates exercises produced significant improvements on joint flexibility, whether performed 5 or 10 times with a 5 sec interval. Additionally, improvements in joint flexibility did not appear to be dependent on the number of repetitions, but most likely on the joint ROM. Thus, mat Pilates exercises, when performed in a full ROM, even in a low number of repetitions, could consist of an effective alternative training method for improving flexibility among amateur soccer players.