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• Supplement of beef extract improves chemotherapy-induced fatigue and toxic effects. • Beef extract improves chemotherapy-fatigue was related to enhance glycogen containing. • Beef extract improves chemotherapy-toxic effect was related with anti-oxidative enzyme activity. Physiological fatigue and toxic effects are commonly associated with chemotherapy. Beef extract (BE) is a nutritional supplement obtained by cooking beef. The present study examined the effects of several BE concentrations on 5-fluorouracil (5-FU)-induced fatigue and toxic effects in mice. Results showed BE supplementation ameliorated 5-FU-induced body and organ weight loss. All BE + 5-FU groups demonstrated increased grip strength (135.3–163.2 g), endurance time (567.3–911.3 s), and movement distance (23623–31225 cm) when compared with the 5-FU group (97.6 g, 265.2 s, and 13916 cm). Hepatic and muscular (0.76 and 1.06 mg/g) glycogen levels were maintained in the BE-3x + 5-FU group when compared with the 5-FU group (0.44 and 0.58 mg/g). Moreover, catalase (liver and muscle) and glutathione peroxidase (liver) activities were enhanced. The serum alanine aminotransferase, aspartate aminotransferase, and histopathological changes were ameliorated in the BE-2x + 5-FU and BE-3x + 5-FU groups. BE supplementation could provide a novel strategy for managing 5-FU-induced fatigue and toxic effects. [ABSTRACT FROM AUTHOR]