The primary aim of the current study is to estimate the effect of different physical activity levels on hair trace element content in male and female students. A total of 113 students (59 women and 54 men) of P. G. Demidov Yaroslavl State University (Yaroslavl, Russia) took part in the current investigation. According to the level of the physical activity, all students were divided into three groups: high, medium, and low physical activity. Essential and toxic metal content (μg/g) in hair samples was assessed by inductively coupled plasma mass spectrometry using NexION 300D + NWR213 (Perkin-Elmer, USA). The obtained data show that hair iodine, zinc, arsenic, nickel, and tin levels are not related to physical activity in male and female students. At the same time, increased physical activity is associated with decreased hair copper, vanadium, bismuth, and mercury content in comparison to the low physical activity groups. Students with higher physical activity are also characterized by significantly higher hair cobalt, iron, manganese, selenium, cadmium, lithium, and lead concentrations. Finally, statistical analysis has revealed maximal gender differences in hair trace element content in the high physical activity groups, whereas in the low activity groups, the hair metal concentrations were nearly similar in females and males.