The aim of this in vitro study was to compare the cyclic fatigue resistance of three single-use nickel-titanium (NiTi) instruments (One Shape, Reciproc and Wave One) after immersion in sodium hypochlorite (NaOCl) solution over several time periods. A total of 135 One Shape, Reciproc R25 and Wave One Primary were tested. The 45 files of the same brand were randomly assigned to three groups (n = 15) and submitted to the following immersion protocol: 1 minute in 5% NaOCl at 37ºC, 5 minutes in 5% NaOCl at 37ºC, no immersion (as control). Cyclic fatigue test was performed rotating/reciprocating all instruments in a stainless steel curved artificial canal (60º angle of curvature and 5 mm radius). The number of cycles to fracture (NCF) was determined by measuring the time to fracture. The data were compared for differences by using 2-way analysis of variance (p < 0.05). In general, resistance to cyclic fatigue was not significantly affected by immersion in NaOCl. Reciproc R2 showed the highest cyclic fatigue resistance in all groups compared to One Shape and Wave One. Immersion in NaOCl for 1 or 5 minutes did not reduce the cyclic fatigue resistance of NiTi single-use instruments significantly. Reciproc were more resistant, but the new rotary single-use One Shape instruments showed mechanical resistance similar to NiTi files developed for reciprocating motion.