This study aimed to compare different thresholds obtained from the muscle near-infrared spectroscopy (NIRS), heart rate variability (HRV), HR and ventilatory parameters during a maximal incremental cycling test. Twenty trained male cyclists performed an incremental step test with breath-by-breath gas analysis, muscle NIRS-derived signal, HRV and HR continuously recorded. The first and second ventilatory thresholds (VT1 and VT2, respectively) were identified and compared to the other methods. The DMAX method was used to determine the NIRS-derived thresholds by oxygenated hemoglobin (O2HbT), deoxygenated hemoglobin (HHbT), tissue saturation index (TSIT); HRV-derived thresholds by standard deviation of the instantaneous (SD1T) and continuous long-term RR intervals (SD2T) and HR deflection point. No significant differences were found between VT1 and O2HbT (171 ± 25 vs 179 ± 46 W; SEE = 23.0 W), VT1 and SD1T (171 ± 39 W; SEE = 26.2 W) and VT1 and SD2T (185 ± 37 W; SEE = 21.7 W); as well as between VT2 and TSIT (242 ± 28 vs 230 ± 61 W; SEE = 25.1 W), when expressed as power-output, respectively. The standard error of estimate for these comparisons is approximately equivalent to one incremental stage (25 W). However, agreement analysis showed substantial inter-individual error (LoA up to ±107 W). This wide variability limits their precision for individual prescription, suggesting these methods should complement rather than substitute ventilatory thresholds.