Background Near-infrared spectroscopy (NIRS) measurements of oxygenation reflectO2 delivery and utilization in exercising muscle and may improve detection of a critical exercise threshold. Purpose First, to detect an oxygenation breakpoint ($Δ$[O2HbMb-HHbMb]-BP) and compare this breakpoint to ventilatory thresholds during a maximal incremental test across sexes and training status. Second, to assess reproducibility of NIRS signals and exercise thresholds and investigate confounding effects of adipose tissue thickness on NIRS measurements. Methods Forty subjects (10 trained male cyclists, 10 trained female cyclists, 11 endurance trained males and 9 recreationally trained males) performed maximal incremental cycling exercise to determine $Δ$[O2HbMb-HHbMb]-BP and ventilatory thresholds (VT1 and VT2). Muscle haemoglobin and myoglobin O2 oxygenation ([HHbMb], [O2HbMb], SmO2) was determined in m. vastus lateralis. $Δ$[O2HbMb-HHbMb]-BPwas determined by double linear regression. Trained cyclists performed the maximal incremental test twice to assess reproducibility. Adipose tissue thickness (ATT) was determined by skinfold measurements. Results $Δ$[O2HbMb-HHbMb]-BPwas not different from VT1, but only moderately related (r = 0.58- 0.63, p<0.001). VT1 was different across sexes and training status, whereas $Δ$[O2HbMb-HHbMb]-BP differed only across sexes. Reproducibilitywas high for SmO2 (ICC = 0.69- 0.97), $Δ$[O2HbMb-HHbMb]-BP (ICC = 0.80-0.88) and ventilatory thresholds (ICC = 0.96- 0.99).SmO2 at peak exercise and at occlusion were strongly related to adipose tissue thickness (r2 = 0.81, p<0.001; r2 = 0.79, p<0.001). Moreover, ATT was related to asymmetric changes in $Δ$[HHbMb] and $Δ$[O2HbMb] during incremental exercise (r = -0.64, p<0.001) and during occlusion (r = -0.50, p<0.05). Conclusion Although the oxygenation threshold is reproducible and potentially a suitable exercise threshold, VT1 discriminates better across sexes and training status during maximal stepwise incremental exercise. Continuous-wave NIRS measurements are reproducible, but strongly affected by adipose tissue thickness.