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We propose a novel method for estimation of muscle fiber conduction velocity from surface electromyographic (EMG) signals. The method is based on the regression analysis between spatial and temporal frequencies of multiple dips introduced in the EMG power spectrum through the application of a set of spatial filters. This approach leads to a closed analytical expression of conduction velocity as a function of the auto- and cross-spectra of monopolar signals detected along the direction of muscle fibers. The performance of the algorithm was compared with respect to that of the classic single dip approach on simulated and experimental EMG signals. The standard deviation of conduction velocity estimates from simulated single motor unit action potentials was reduced from 1.51 m/s [10 dB signal-to-noise ratio (SNR)] and 1.06 m/s (20 dB SNR) with the single dip approach to 0.51 m/s (10 dB) and 0.23 m/s (20 dB) with the proposed method using 65 dips. When 200 active motor units were simulated in an interference EMG signal, standard deviation of conduction velocity decreased from 0.95 m/s (10 dB SNR) and 0.60 m/s (20 dB SNR) with a single dip to 0.21 m/s (10 dB) and 0.11 m/s (20 dB) with 65 dips. In experimental signals detected from the abductor pollicis brevis muscle, standard deviation of estimation decreased from (mean plusmn SD over 5 subjects) 1.25 plusmn 0.62 m/s with one dip to 0.10 plusmn 0.03 m/s with 100 dips. The proposed method does not imply limitation in resolution of the estimated conduction velocity and does not require any iterative procedure for the estimate since it is based on a closed analytical formulation.