Background/Objectives: To evaluate the concentrations of trace elements in tear fluid among athletes using particle-induced X-ray emission (PIXE), and to assess the associations with gender, sports intensity, and nutritional supplement intake. Methods: In this cohort study, 84 athletes engaged in high- or low-intensity sports completed a demographic and supplement-use questionnaire. Tear samples were collected using Schirmer strips and analyzed for elemental composition with PIXE, a high-sensitivity technique suited for small biological samples. Multivariate and nonparametric statistical analyses were used to compare groups. Results: There were 46 males and 38 females, aged 17–63 years (mean 30.21 years). Tear phosphorus, potassium, and sulfur concentrations were higher in women than men and higher in women participating in low-intensity compared to high-intensity sports. Tear concentrations of magnesium were higher in men participating in high-intensity sports compared to low-intensity sports. They were higher in men than women regardless of supplement intake. Iron concentrations were higher in men than women only when neither group was taking supplements. Smoking had a slight inverse relationship to iron values. Iron levels were particularly high in men participating in intense sports and low in smokers. Magnesium supplements were associated with raised magnesium levels in tears. Conclusions: This study demonstrates an association between trace element levels in human tears and gender, sports intensity, and food supplement intake. PIXE enables the evaluation of trace element concentration in tears, which may serve as potential biomarkers for the clinical assessment of athletes’ health.