This study was designed to assess whether symptoms, functional measures, and reported disabilities were associated with vitamin B12 (B12) deficiency when defined in three ways. Participants, aged 60 or more years of age, in 1999–2002 National Health and Nutrition Examination Surveys (NHANES) were categorized in relation to three previously used definitions of B12 deficiency: (1) serum B12 < 148 pmol/L; (2) serum B12 < 200 pmol/L and serum homocysteine > 20 μmol/L; and (3) serum B12 < 258 pmol/L or serum methylmalonic acid > 0.21 μmol/L. Functional measures of peripheral neuropathy, balance, cognitive function, gait speed, along with self-reported disability (including activities of daily living) were examined with standardized instruments by trained NHANES interviewers and technicians. Individuals identified as B12 deficient by definition 2 were more likely to manifest peripheral neuropathy OR (odds) (95% confidence intervals), p
value: 9.70 (2.24, 42.07), 0.004 and report greater total disability, 19.61 (6.22, 61.86) 0.0001 after adjustments for age, sex, race, serum creatinine, and ferritin concentrations, smoking, diabetes, and peripheral artery disease. Smaller, but significantly increased, odds of peripheral neuropathy and total disability were also observed when definition 3 was applied. Functional measures and reported disabilities were associated with B12 deficiency definitions that include B12 biomarkers (homocysteine or methylmalonic acid). Further study of these definitions is needed to alert clinicians of possible subclinical B12 deficiency because functional decline amongst older adults may be correctable if the individual is B12 replete.