Abstract Objectives To explore patients’ use of patient portals to access lab test results, their comprehension of lab test data, and factors associated with these. Materials and Methods An online survey was administered to 276 adults 18+ years. Multivariate logistic regression was used to determine factors associated with patient portal use to view lab test results and lab test comprehension. Results The sample was predominantly White (72.5%), female (55.4%), with mean age 50.7 ± 15.5 years. Overall, participants had low numeracy (10.79 ± 2.71) and eHealth literacy skills (23.91 ± 5.29), and moderate lab test comprehension scores (18 ± 2.3). White participants with greater eHealth literacy were more likely to access lab test results via patient portals, whereas those with a college education were less likely to do so (χ2 [3]=31.23, P < .001). The regression showed that older age, fewer chronic conditions, and use of patient portals were significantly related to higher lab test comprehension scores (F [22, 250]=8.73, P <.001). Older adults performed better on comprehension tests but reported having difficulty understanding lab tests, expressing a preference for their doctors to explain them. Discussion The findings shed light on the experiences and needs of different user groups that must be addressed to enhance their ability to effectively use patient portals for obtaining lab test results. Conclusion More research is needed to determine patient barriers to comprehending lab test results online and to develop tailored strategies to improve patients’ self-efficacy to meaningfully use medical information in patient portals.