Recently, a new method has been proposed to detect the onset of neuromuscular fatigue during an incremental cycling test by assessing the changes in spectral electromyographic (sEMG) frequencies within individual exercise periods of the test. The method consists on determining the highest power output that can be sustained without a significant decrease in spectral frequencies. This study evaluated the validity of the new approach by assessing the changes in spectral indicators both throughout the whole test and within individual exercise periods of the test. Fourteen cyclists performed incremental cycle ergometer rides to exhaustion with bipolar surface EMG signals recorded from the vastus lateralis. The mean and median frequencies (Fmean and Fmedian, respectively) of the sEMG power spectrum were calculated. The main findings were: (1) Examination of spectral indicators within individual exercise periods of the test showed that neither Fmean nor Fmedian decreased significantly during the last (most fatiguing) exercise periods. (2) Examination of the whole incremental test showed that the behaviour of Fmean and Fmedian with increasing power output was highly inconsistent and varied greatly among subjects. (3) Over the whole incremental test, half of the participants exhibited a positive relation between spectral indicators and workload, whereas the other half demonstrated the opposite behavior. Collectively, these findings indicate that spectral sEMG indexes do not provide a reliable measure of the fatigue state of the muscle during an incremental cycling test. Moreover, it is concluded that it is not possible to determine the onset of neuromuscular fatigue during an incremental cycling test by examining spectral indicators within individual exercise periods of the test.