Objective The level of skeletal muscle glucose metabolism is an important factor affecting aerobicexercise capacity, and is regulated by the biological clock. Chrono is a newly discovered circadian clock gene. It isinvolvedin the feedback inhibition of transcription of the core circadian clock transcription factor BMAL1 to its downstream circadianclock target genes, and is considered a transcription repressor of BMAL1. Studies have shown that BMAL1 is involved inthe regulation of skeletal muscle glucose metabolism. However, CHRONO is a transcription inhibitor of BMAL1, and theeffect of the CHRONO-BMAL1 pathway on exercise capacity and glucose tolerance is currently unknown. Therefore, thisstudy employed skeletal muscle-specific Chrono overexpression mice and wild-type mice to explore the effect of theCHRONO-BMAL1 pathway on glucose tolerance and exercise capacity. Our findings provide a theoretical basis forelucidating new mechanisms affecting skeletal muscle health. Methods In total, 20 healthy 8-week-old C57BL/6N wildtype(WT) mice and 20 skeletal muscle-specific Chrono overexpression (Chrono-TG) mice (50:50, male: female) wereused in this study. Body weight and food intake were recorded. The body composition test, autonomous activity test,incremental load exercise ability test, glucose tolerance test, and grip test were performed. Skeletal muscle was weighedand the mRNA expression levels of the Chrono, MyhcI, MyhcIIa, MyhcIIb, MyhcIIx, and Pdha1 genes were detected byReal-time PCR. The content of muscle glycogen was detected using an appropriate kit. Results (1) Compared with WTmice, the autonomous mobility and exercise ability of Chrono-TG mice were obviously reduced ( P < 0. 05 or P < 0. 01);(2) Compared with WT mice, the weights of the gastrocnemius, quadriceps, soleus, extensor digitorum longus, and tibialanterior muscles of TG mice were significantly increased ( P < 0. 01), while the weights of the gastrocnemius andquadriceps femoris of female TG mice were lower than for male TG mice ( P < 0. 01); (3) Compared with WT mice, thestrength of the front paw of male TG mice was significantly reduced ( P < 0. 05), and MyhcIIs mRNA expression in skeletalmuscle was also decreased or showed a downward trend. The forepaw grip and MyhcIIs mRNA expression in female TG micewere clearly higher than in male TG mice ( P < 0. 05 or P < 0. 01); (4) Compared with WT mice, the blood glucose valueand the area under the curve at each point in the glucose tolerance test of the TG mice were increased ( P < 0. 01), whilefemale TG mice showed lower levels than male TG mice ( P < 0. 01); (5) Compared with WT mice, the content of muscleglycogen in TG mice was increased ( P < 0. 05), and the expression of Pdha1 mRNA was decreased or showed a decreasingtrend, while the muscle glycogen content and Pdha1 mRNA expression in female TG mice were significantly higher than inmale TG mice ( P < 0. 05 or P < 0. 01). Conclusions Overexpression of the Chrono gene in skeletal muscle can reduceautonomic mobility, impair glucose tolerance, and affect the aerobic exercise capacity of mice.