The objective of this study was to assess the electromyographic activity of the rectus abdominis (upper and lower part) and external oblique during sit-ups performed on BOSU ball(s). Twenty-four men participated in a familiarization session, and in the next session, they performed the experimental tests in randomized order. The sit-ups were performed with 10 repetitions with body weight and with 10 repetition maximum (10RM) using elastic bands as external resistance under 4 different conditions: (a) on a stable surface, (b) with the BOSU ball under their feet (dome side down, lower-body instability), (c) BOSU ball under the low back (dome side up, upper-body instability), and (d) with BOSU balls under both feet and the low back (dual instability). The feet were not attached to the surface. We observed that with body weight, external oblique activation was decreased by upper-body instability and dual instability by 22-24% (p = 0.002-0.006), whereas the rectus abdominis was not affected by the surface. Using 10RM loads, the upper and lower rectus abdominis activities were increased by upper body and dual instability by 21-24% compared with that for a stable surface (P ≤ 0.001-0.036). Further, lower-body instability did not affect muscle activities significantly with either load for any condition. Hence, BOSU balls under the low back can increase and decrease abdominal muscle activation depending on the load, whereas placing a BOSU ball under the feet with the dome side down had little impact.