The aim was to assess the trace element contents in toenails of older adults and its association with regular physical activity. Cross-sectional multicentre study in Spain, collecting data from a random sample of 380 participants (54% female) aged 55-80 years (men) and 60-80 years (women) with no previously documented cardiovascular disease. Physical activity performed was measured using the Minnesota Leisure-time Physical Activity Questionnaire. The 25 most inactive and 25 most active individuals for each sex were selected for this study (final sample n = 100). Anthropometric measurements were performed and toenail samples collected for calcium (Ca), chromium (Cr), iron (Fe), cobalt (Co), nickel (Ni), zinc (Zn), selenium (Se) and mercury (Hg) analysis. Significant differences between sexes were reported in Ca concentrations, women having lower concentrations than men. No differences were reported in trace element contents between active and inactive men. Active women showed higher Ca, Cr, Fe, Co, and Zn and lower Hg contents than their inactive peers (all p<0.05). Inactive women showed lower Ca and Co levels (735.0 mg/kg and 4.5 μg/kg, respectively) than inactive men (1170.0 mg/kg and 7.9 μg/kg, respectively). Active women had lower Ca and higher levels of Cr (936.0 mg/kg and 1230.0 μg/kg, respectively) than active men (1070.0 mg/kg and 522.0 μg/kg, respectively). The present data added new information on the element contents in toenails of healthy Spanish older adults. The concentration of trace elements was similar in both sexes except for Ca which were lower in women. The trace element contents in women's toenails, but not in men, were markedly influenced by physical activity, with higher levels of Ca and Fe and lower Hg among active females.