Slettaløkken, G and Rønnestad, BR. High-intensity interval training every second week maintains V[Combining Dot Above]O2max in soccer players during off-season. J Strength Cond Res 28(7): 1946–1951, 2014—Reduced endurance training among semiprofessional soccer players during off-season may have negative effect on game performance during the competition season. This negative effect can be prevented by adding high-intensity interval training (HIT) to normal activity. In this study, we want to compare 2 different frequencies of HIT (5 bouts of 4 minutes on 87–97% peak heart rate) session on maintenance of aerobic fitness among semiprofessional soccer players during a 6-week off-season period. Seventeen male players at second and third highest soccer division in Norway participated. The subjects were randomized into 1 HIT session every second week (HIT 0.5) or 1 HIT session per week (HIT 1). All participants performed a 20-m shuttle run test and a maximal oxygen uptake (V[Combining Dot Above]O2max) test on treadmill before and after the training intervention. V[Combining Dot Above]O2max (HIT 0.5, 63.4 ± 5.9 ml·kg−1·min−1; HIT 1, 65.6 ± 2.1 ml·kg−1·min−1) and 20-m shuttle run performance (HIT 0.5, 2335 ± 390 m, HIT 1, 2531 ± 106 m) were not different between the groups before the training intervention. V[Combining Dot Above]O2max was maintained after the training intervention in both HIT 0.5 and HIT 1 (64.0 ± 5.9 ml·kg−1·min−1and 64.3 ± 1.3 ml·kg−1·min−1, respectively). There was a reduction in distance covered during the 20-m shuttle run test in HIT 1 and when groups were pooled (−7.9 ± 5.7% and −6.4 ± 7.9%, respectively, p ≤ 0.05). In conclusion, HIT 1 did not maintain V[Combining Dot Above]O2max better than HIT 0.5 when added to normal off-season activity. However, performance in 20-m shuttle run, which is a more soccer-specific fitness test than V[Combining Dot Above]O2max test, was slightly reduced when both groups was pooled.