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Electromyographic (EMG) recordings detected over the skin may be mixtures of signals generated by different active muscles due to the phenomena related to volume conduction. Separation of the sources is necessary when single muscle activity has to be detected. Signals generated by different muscles may be considered uncorrelated but in general overlap in time and frequency. Under certain assumptions, mixtures of surface EMG signals can be considered as linear instantaneous but no a priori information about the mixing matrix is available when different muscles are active. In this study, we applied blind source separation (BSS) methods to separate the signals generated by two active muscles during a force-varying task. As the signals are non stationary, an algorithm based on spatial time-frequency distributions was applied on simulated and experimental EMG signals. The experimental signals were collected from the flexor carpi radialis and the pronator teres muscles which could be activated selectively for wrist flexion and rotation, respectively. From the simulations, correlation coefficients between the reference and reconstructed sources were higher than 0.85 for signals largely overlapping both in time and frequency and for signal-to-noise ratios as low as 5 dB. The Choi-Williams and Bessel kernels, in this case, performed better than the Wigner-Ville one. Moreover, the selection of time-frequency points for the procedure of joint diagonalization used in the BSS algorithm significantly influenced the results. For the experimental signals, the interference of the other source in each reconstructed source was significantly attenuated by the application of the BSS method. The ratio between root-mean-square values of the signals from the two sources detected over one of the muscles increased from (mean ± standard deviation) 2.33±1.04 to 4.51±1.37 and from 1.55±0.46 to 2.72±0.65 for wrist flexion and rotation, respectively. This increment was statistically significant. It was concluded that the BSS approach applied is promising for the separation of surface EMG signals, with applications ranging from muscle assessment to detection of muscle activation intervals, and to the control of myoelectric prostheses.