Biological variation (BV) is intertwined with many considerations in laboratory medicine and forms a basis for setting analytical performance specifications, delta check parameters, reference intervals, and reference change values. Biological variation describes the fluctuation in analyte concentration around a homeostatic setpoint within a single subject (within-subject BV) or a group of subjects (between-subject BV). Literature evaluating BV data in laboratory medicine stretches back over 50 years; however, the lack of standardization and appropriate study designs to derive BV data has led to significant discrepancy in reported BV estimates. This lack of reproducibility in older BV studies significantly hindered its adoption into clinical practice.