The study aimed to investigate the interaction of vitamin D and calcium with high-intensity circuit training on BDNF and fat percent in overweight elderly. Thirty-two healthy older women randomly divided into four groups: 1. Training+supplementation, 2. Training+placebo, 3. Supplementation, and 4. Control. The training performed three sessions/week for eight weeks. The Supplementation groups consumed 1,000 mg of effervescent calcium daily and 50,000 IU of vitamin D per week. The results showed that the body fat percent significantly decreased in training+supplementation group compared to supplementation (p = 0.001), control (p = 0.001) and training+placebo (p = 0.045) groups. Moreover, fat percent significantly decreased in the training+placebo group compared to the control group (p = 0.001). However, there were no significant differences between the training+placebo and supplementation groups in body fat percent. Moreover, there was a significant decrease in fat percent in the supplementation group compared to the control group (p = 0.047). BDNF in the training+supplementation group had a significant increase compared to the supplementation (p = 0.002) and control groups (p = 0.001) but there was no significant difference compared to the placebo+training (p = 0.085). Moreover, there was a significant increase in BDNF in the training+placebo compared to control (p = 0.003) and supplementation (p = 0.01) groups. In contrast, no significant difference between supplementation and control (p = 0.985) groups was observed. Overall, the interaction of training and supplementation showed a significant decrease in the body fat percentage and a significant increase in BDNF in overweight elderly.