Background Commutable reference materials (RMs) are suitable for end-users for evaluating the metrological traceability of values obtained using routine measurement systems. We assessed the performance of 6 routine measurement systems with validated secondary RMs. Methods We tested the homogeneity, stability, and commutability of 5 minimally processed human serum pools according to the standard guidelines. The serum pools were assigned values as per the reference procedure of the United States Centers for Disease Control and were used to evaluate the trueness of results from 6 commercial measurement systems based on enzymatic methods: 3 glucose oxidase (GOD) and 3 hexokinase (HK) methods. Results The prepared RMs were validated to be sufficiently homogenous, stable, and commutable with the patient samples. Method bias varied for different systems: GOD01, -0.17 to 2.88%; GOD02, 1.66 to 4.58%; GOD03, -0.17 to 3.14%; HK01, -3.48 to -0.85%; HK02, -3.83 to -0.11%, and HK03, -1.82 to -0.27%. Conclusions We observed that the prepared serum glucose RMs were qualified for trueness assessment. Most of the measurement systems met the minimal quality specifications.