Background: Physical exercise and the subsequent recovery processes have been proposed to affect the essential hemostatic balance of trace elements, either by increasing degradation rates or by enhancing loss from the body. This might disturb the function of vital metabolic organs, such as muscles and liver, leading to unsatisfactory performance. It appears that taking advantage of mineral-rich herbal supplements could replenish the body's mineral sources. Objectives: The present study aimed to investigate the effects of Cucurbita pepo supplementation on exercise-induced dynamics of micronutrients in serum, liver, and skeletal muscle of Wistar rats. Methods: Forty male Wistar rats (aged 4 - 6 weeks old, weighing 130 - 150 g) were randomly divided into four groups of ten rats each: (1) Saline control group (SC), (2) training at 25 m/min group (ST), (3) pumpkin seed oil supplementation group (PC), and (4) pumpkin seed oil supplementation + Training at 25m/min (PT). Moderate-intensity aerobic exercise (25 m/min, eliciting approximately 65% VO2Peak) and consumption of pumpkin seed oil (4 ml/kg/day) were conducted for 6 weeks. The ICP-OES technique was used to measure plasma, liver, and skeletal muscle levels of zinc (Zn), iron (Fe), manganese (Mn), magnesium (Mg), and copper (Cu) approximately 48 to 72 hours after the final session of the training protocol. Data were analyzed using one-way ANOVA and post-hoc Bonferroni tests (P < 0.05). Results: No significant changes in serum Fe (P = 0.728) and Mn (P = 0.52) levels, as well as liver tissue levels of Zn (P = 0.633), Fe (P = 0.512), Mn (P = 0.194), Mg (P = 0.156), and Cu (P = 0.711), were observed among the intervention groups. However, all trace mineral element (TME) concentrations, except for Cu (P = 0.907) in the gastrocnemius tissue, significantly changed among the study groups (P < 0.05). Additionally, the results of the Bonferroni test showed that six weeks of moderate physical activity decreased the serum, liver, and gastrocnemius levels of TMEs, and subsequent supplementation with pumpkin seed oil played a replenishing role in preserving the resources. Conclusions: Pumpkin seed oil supplementation was demonstrated to be an effective ergogenic aid for the up-regulation and replenishment of exercise-induced TME excretion from the liver, gastrocnemius, and body fluids in healthy rats.