We conducted a pilot study in adults with and without type 2 diabetes (T2D), measuring whole-blood thiamine diphosphate (TDP) concentrations alongside erythrocyte transketolase activity (TKT) and activity coefficients (ETKAC), and evaluated these findings alongside dietary and ETKAC data from the U.K. National Diet and Nutrition Survey (NDNS). In the pilot cohort, circulating TDP concentrations did not differ between T2D and control groups, and ETKACs were largely sufficient, yet absolute TKT activities varied approximately sixfold among participants. Magnesium supplementation of erythrocyte lysates increased TKT activity, demonstrating that cofactor availability beyond TDP can influence functional measurements. Discordance between circulating TDP and functional status was also observed, including ETKAC-defined deficiency despite a TDP concentration within the reference range. Among participants with T2D, TDP was positively associated with fasting glucose and HbA1c, a relationship absent in controls. In NDNS analyses, higher thiamine intake and dietary density were strongly associated with lower ETKAC-defined non-sufficiency, although substantial non-sufficiency persisted despite adequate intake. Dietary thiamine density, ethnicity, age, and magnesium intake were independently associated with ETKAC status. Collectively, these findings support the conclusion that circulating TDP, dietary intake, and ETKAC provide related, but non-equivalent measures and support integrating both direct and functional assessments of thiamine status.