OBJECTIVES/GOALS: We developed a non-invasive test of ocular alignment (OA) as a measure of otolith asymmetry, which impacts vestibular function. This test has also been administered on commercial astronauts to study the effect of microgravity on vestibular function. We will assess OA test performance in ground subjects, which has not yet been characterized. METHODS/STUDY POPULATION: Subjects were recruited from visitors to Space Center Houston from June to October 2022. Participants wore red-blue glasses in a darkened room and aligned two vertically or torsionally misaligned line segments, one red and one blue, on a tablet. Each subject underwent 11 vertical and torsional trials. The remaining misalignment was used to calculate ocular misalignment. Subjects self-reported demographic and health data. We calculated descriptive statistics and explored the distribution of overall vertical and torsional OA and distributions by age. Next steps will establish the reliability of the outcome measures with intraclass correlation coefficients and analyze associations between demographic variables and health status with OA performance using linear regression analysis. RESULTS/ANTICIPATED RESULTS: A total of 143 participants (67 female) with a median age of 21 (range 5-74 years old) completed OA testing. Of participants, 64% identified as white, 10% as black, 14% as Asian, 3% as Native American, and 8% as unknown. This test battery was feasible to implement in this mixed-age sample, with a 95% completion rate. Median absolute vertical OA was 0.08 degrees (IQR = 0.17 degrees), and median absolute torsional OA was 0.80 degrees (IQR = 1.20 degrees). Visual analysis of plots of OA by trial number reveals no apparent learning effect within subjects, though some individuals had considerable variation in performance. Average absolute vertical ocular misalignment increased modestly when plotted by age (fitted regression line b = 0.0032 degrees per year of age, p = 0.004), unadjusted for potential confounders. DISCUSSION/SIGNIFICANCE: Ocular alignment testing was feasible in a mixed-age general population sample. Our findings will expand understanding of how OA varies with age. These results will be used to create a normative database to compare and inform analysis of OA results from commercial spaceflights, which include individuals of varying age and health statuses.