C₆₀ fullerene (C₆₀) as a nanocarbon particle, compatible with biological structures, capable of penetrating through cell membranes and effectively scavenging free radicals, is widely used in biomedicine. A protective effect of C₆₀ on the biomechanics of fast (m. gastrocnemius) and slow (m. soleus) muscle contraction in rats and the pro- and antioxidant balance of muscle tissue during the development of muscle fatigue was studied compared to the same effect of the known antioxidant N-acetylcysteine (NAC). C₆₀ and NAC were administered intraperitoneally at doses of 1 and 150 mg kg⁻¹, respectively, daily for 5 days and 1 h before the start of the experiment. The following quantitative markers of muscle fatigue were used: the force of muscle contraction, the level of accumulation of secondary products of lipid peroxidation (TBARS) and the oxygen metabolite H₂O₂, the activity of first-line antioxidant defense enzymes (superoxide dismutase (SOD) and catalase (CAT)), and the condition of the glutathione system (reduced glutathione (GSH) content and the activity of the glutathione peroxidase (GP_x) enzyme). The analysis of the muscle contraction force dynamics in rats against the background of induced muscle fatigue showed, that the effect of C₆₀, 1 h after drug administration, was (15–17)% more effective on fast muscles than on slow muscles. A further slight increase in the effect of C₆₀ was revealed after 2 h of drug injection, (7–9)% in the case of m. gastrocnemius and (5–6)% in the case of m. soleus. An increase in the effect of using C₆₀ occurred within 4 days (the difference between 4 and 5 days did not exceed (3–5)%) and exceeded the effect of NAC by (32–34)%. The analysis of biochemical parameters in rat muscle tissues showed that long-term application of C₆₀ contributed to their decrease by (10–30)% and (5–20)% in fast and slow muscles, respectively, on the 5th day of the experiment. At the same time, the protective effect of C₆₀ was higher compared to NAC by (28–44)%. The obtained results indicate the prospect of using C₆₀ as a potential protective nano agent to improve the efficiency of skeletal muscle function by modifying the reactive oxygen species-dependent mechanisms that play an important role in the processes of muscle fatigue development.