The first evidence of unwanted serum protein effects on analogue-based total thyroxine (T4) determinations came from a study that varied serum protein concentrations while total T4 concentrations were constant. The present study approached this issue by varying total T4 concentrations while protein concentrations were constant. Four analogue-based total T4 immunoassays were applied to solutions that contained either free T4 without binding protein, a T4-binding protein without T4, or protein-bound T4. When total T4 concentrations were 3-12 microg/dL, the assays reported total T4 determinations that ranged from none detected to 23 microg/dL. These T4 determinations reflected the protein to which T4 was bound, in addition to the level of T4. Total T4 was underrepresented when T4 was unbound, or thyroxine-binding globulin (TBG) bound. Total T4 was overrepresented when T4 was albumin-bound, or transthyretin-bound. There were substantial disparities among assays applied to the same total T4 solutions. These assays reported no detectable T4 when applied to T4-binding protein solutions without protein-bound T4. Nonetheless, T4-binding proteins contributed to the underestimates and overestimates of protein bound T4. Different forms of protein bound T4 were quantified differently, evidence that protein-T4 complexes persist during quantification. We attribute the unexpected total T4 values to a combination of incomplete protein-bound T4 release from T4-binding proteins during quantification, and variably inaccurate quantifications of the protein-bound T4 that remained.