Abstract Exposure to toxic metals and metalloids is a significant public health concern and can seriously affect human health. The aim of this study was to establish the presence of toxic metals and metalloids in the urine of non‐professional sportsmen who participated in a mountain ultramarathon using a benchtop total reflection x‐ray fluorescence (TXRF) system. TXRF allows for easy and rapid sample preparation and is therefore a potential candidate for simple and cost‐effective analysis. In this work, the TXRF‐Mo system was used for multielement analysis of the urine samples of 21 non‐professional athletes who ran a 53 km mountain ultra‐marathon. Urine samples were collected at four time points, at the beginning of the race (pre‐race samples), immediately, and 12 and 24 h post‐race. Al, As, Ba, Ni, Pb, Rb, Sr, V, and Tl were detected in the collected urine samples. The accuracy and precision of the proposed methods were verified by the analysis of reference materials (Seronorm™ Trace Elements Urine Level 2). The TXRF results were in agreement with the reference values and no significant differences were observed at the 95% confidence level. The detection limits for the elements of interest were also reasonable considering their concentration ranges in real samples. Changes were observed over time with increasing average urinary metals and metalloids levels, but only two significant results, were an increase in As and Rb. The results indicate a high degree of inter‐subject variability. The results obtained show that the content of toxic metals and metalloids increases in the urine samples collected after the race, which could confirm the statement that physical activity can increase the excretion of toxic metals and metalloids from the body. The simplicity of the TXRF method as well as its fast performance make it suitable for routine analysis.