Purpose: To evaluate the effect of age, gender and body mass index (BMI) on the levels of tear film neuromediators and corneal nerve parameters in healthy individuals.Methods: Twenty-six healthy subjects were screened for any neurological deficits. The concentration of substance P and calcitonin gene-related peptide (CGRP) in tears was measured by enzyme-linked immunosorbent assay. Corneal nerve fibers were imaged using confocal microscopy and assessed by automated image analysis software. Associations between the clinical variables were analyzed using Pearson or Spearman correlation. Univariate general linear regression was performed to examine the independent relationship between age, BMI and gender of the subjects with concentrations of substance P, CGRP and corneal nerve fiber parameters.Results: Fifteen (58%) of the study participants were male. The mean age of the study cohort was 36 ± 12 years (range, 21-59) with an average BMI of 25 ± 4 kg/m2. The median [IQR] concentrations of substance P and CGRP was 715 [372-1463] pg/mL and 38 [15-74] ng/mL respectively. Moderate but significant positive correlations were found between the concentration of substance P and corneal nerve fiber density (r = 0.467, P = .016), nerve fiber length (r = 0.528, P = .006) and nerve fractal dimension (rs = 0.614, P = .002). There was a significant age-dependent reduction in the concentration of substance P (-6% pg/mL per year, P = .001) and CGRP (-8% ng/ml per year, P < .001). Corneal nerve fiber density (-0.171 no./mm2 per year, P = .029) and nerve fractal dimension (-0.001 per year, P = .021) showed reductions with advancing age. Gender and BMI did not influence any of the measurements.Conclusions: The concentrations of substance P and CGRP in tears, as well as corneal nerve fiber density and nerve fractal dimension, are significantly reduced with advancing age. Age should be considered when evaluating patients for diagnosis and follow-up of corneal neuropathy or ocular surface disorders.