Mitochondria activation factor (MAF) which is high-molecular weight polyphenol contained in black tea and oolong tea can increase the mitochondrial membrane potential. MAF supplementation to mice facilitates endurance running performance after 9-week endurance training and muscle hypertrophy induced by synergist ablation. In this study, we examined the effect of oral MAF supplementation on overall physical fitness (expressed as physical fitness age) in physically active middle-aged and older women. This study is a randomized double-blind placebo-controlled trial implemented between January and May 2019 at three fitness facilities in Ibaraki, Japan. Seventy middle-aged women aged 55 to 69 years were randomly assigned into placebo (n = 35) and MAF groups (n = 35). The placebo participants took cornstarch-containing capsules, and the MAF participants took MAF-containing capsules twice a day for 80 days consecutively. During the intake period, all participants were instructed to follow 30-min circuit training program at least twice a week. Physical fitness age was computed with eight physical fitness items assessing upper-extremity muscle strength, locomotion, and postural change. The physical fitness age decreased by 1.48 years (95% confidence interval [CI]: -2.66, -0.30) in the placebo group and 3.01 years (95% CI: -4.16, -1.86) in the MAF group. The reduction was greater in the MAF group, but did not reach statistical significance (p = 0.06). The combination of 80-day of MAF intake and circuit exercise showed beneficial results. However, our results did not indicate clear effects on physical fitness age because of low statistical power. Further studies are necessary to reveal the effects of MAF supplementation.