Background The fifth generation (high-sensitivity) troponin T assay offers increased precision and analytical sensitivity to the predecessor method. The assay has proven utility in risk stratification and patient management. Upon clinical suspicion and discordant 4th generation troponin T and troponin I results, we investigated a sample for suspected interfering substances to the 5th generation troponin T assay. Methods The analysis included a serial dilution, treatment with polyethylene glycol, commercial antibody blocking reagents, and size exclusion chromatography. Results The sample diluted linearly (R2 = 0.9957); however, experienced a dramatic reduction in concentration after both the polyethylene glycol and blocking agent treatment. Finally, size exclusion chromatography demonstrated assay reactivity around 970 kDa range. Conclusions These experiments elucidate a heterophilic antibody interference to the assay, and demonstrate potential measures to discern the interference.