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Objective: This work was aimed to analyze the effect of vitamin energy drink on muscle fatigue by surface electromyography (SEMG) and ultrasonic bioimaging (USBI). Methods: 20 healthy men were selected to do increasing load fatigue test. Surface electromyographic signals and ultrasonic biological images were collected based on wavelet threshold function with improved thresholds. Time domain and frequency domain characteristic integrated electromyography (IEMG), root mean square amplitude (RMS), average power frequency (MPF), and surface and deep muscle morphological changes were analyzed. Hemoglobin concentration (HB), red blood cell number (RBC), mean volume of red blood cell (MCV), blood lactic acid (BLA), malondialdehyde (MDA), and phosphocreatine kinase (CK) were measured. Results: 1) the Accuracy (94.10%), Sensitivity (94.43%), Specificity (93.75%), and Precision (94.07%) of the long and short-term memory (LSTM) specificity for muscle fatigue recognition were higher than those of other models. 2) Compared with the control group, the levels of BLA, MDA, and CK in the experimental group were decreased and HB levels were increased after exercise (P < 0.05). 3) IEMG and RMS of the experimental group were higher than those of the control group, and increased with time (P < 0.05). 4) The mean amplitude of the response signal decreased with time. Compared with the control group, the surface muscle thickness, deep muscle thickness, total muscle thickness, contrast, and homogeneity (HOM) decreased in the experimental group; while the angular second moment (ASM) and contrast increased, showing great differences (P < 0.05). Conclusion: Surface electromyographic signal and ultrasonic biological image can be used as auxiliary monitoring techniques for muscle fatigue during exercise. Drinking vitamin energy drinks before exercise can relieve physical fatigue to a certain extent and promote the maintenance of muscle microstructure. [ABSTRACT FROM AUTHOR]