In this paper, an attempt has been made to analyze surface electromyography (sEMG) signals under non-fatigue and fatigue conditions using time-frequency based features. The sEMG signals are recorded from biceps brachii muscle of 50 healthy volunteers under well-defined protocol. The pre-processed signals are divided into six equal epochs. The first and last segments are considered as non-fatigue and fatigue zones respectively. Further, these signals are subjected to B-distribution based quadratic time-frequency distribution (TFD). Time frequency based features such as instantaneous median frequency (IMDF) and instantaneous mean frequency (IMNF) are extracted. The expression of spectral entropy is modified to obtain instantaneous spectral entropy (ISPEn) from the time-frequency spectrum. The results show that all the extracted features are distinct in both conditions. It is also observed that the values of all features are higher in non-fatigue zone compared to fatigue condition. It appears that this method is useful in analysing various neuromuscular conditions using sEMG signals. © 2015 World Scientific Publishing Company.