CD4+CD25+FoxP3+ human regulatory TCELLS (TREG) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies, along with first clinical data, indicate TREG to be auspicious to use for future cell therapies, e.g. to induce tolerance after solid organ transplantation. To this end, TREG suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 hour-protocol for the assessment of TREG function by suppression of the early activation markers CD154 and CD69 on CD4+CD25- responder TCELLS (TRESP) upon polyclonal stimulation via anti-CD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall. Our results demonstrate that the observed decrease in activation marker frequency on TRESP is due to competition for anti-CD3/28-coated microbeads as opposed to a TREG-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive TREG function.