Evaluating the accuracy of estimated glomerular filtration rate (eGFR) derived from serum creatinine (SCr) and serum cystatin C (SCysC) equations requires gold-standard measures of GFR. However, the influence of imprecise measured GFRs (mGFRs) on estimates of equation error is unknown.Diagnostic test study.1,995 participants from the Modification of Diet in Renal Disease (MDRD) Study and African American Study of Kidney Disease and Hypertension (AASK) with at least 2 baseline mGFRs from iodine 125–iothalamate urinary clearances, 1 standardized SCr value, and 1 SCysC value.eGFRs calculated using the 4-variable isotope-dilution mass spectrometry (IDMS)-traceable MDRD Study equation, the Chronic Kidney Disease (CKD) Epidemiology Collaboration (CKD-EPI) SCysC equation, the CKD-EPI SCr-SCysC equation, and mGFRs collected from another prerandomization visit.A single reference mGFR, average of 2, and average of 3 mGFRs; additional analysis limited to consistent mGFRs (difference ≤25% from reference mGFR).We found that mGFRs had stable mean values, but substantial variability across visits. Of all mGFRs collected a mean of 62 days apart from the reference visit, 8.0% were outside 30% of the single reference mGFR (1 − P 30 ). Estimation equations were less accurate because 12.1%, 17.1%, and 8.3% of eGFRs from the MDRD Study, CKD-EPI SCysC, and CKD-EPI SCr-SCysC equations were outside 30% of the same gold standard (1 − P 30 ). However, improving the precision of the reference test from a single mGFR to the average of 3 consistent mGFRs decreased these error estimates (1 − P 30 ) to 8.0%, 12.5%, and 3.9%, respectively.Study population limited to those with CKD.Imprecision in gold-standard measures of GFR contribute to an appreciable proportion of the cases in which eGFR and mGFR differ by >30%. Reducing and quantifying errors in gold-standard measurements of GFR is critical to fully estimating the accuracy of GFR estimates.