Objectives/Goals: To assess the association between integrase strand-transfer inhibitor (INSTI) use and sarcopenia, pre-frailty, and frailty in people living with HIV (PLWH) compared to non-INSTI users and HIV-seronegative controls, focusing on changes in muscle strength, function, and physical activity over time. Methods/Study Population: We analyzed data from the MACS/WIHS Combined Cohort Study (CCS) as part of the SPPACE INSTI Study. Eligible participants were PLWH on ART for ≥2 years or individuals at risk for HIV, with exclusions for pregnancy, active tuberculosis, malignancy, or PrEP use. Participants were grouped into 1) INSTI group (PLWH who switched/added INSTI), 2) non-INSTI group, and 3) HIV-seronegative controls. A total of 2,071 women and 1,807 men (2006–2020) were included. Sarcopenia analyses (AIM 1) included participants with grip strength/BMI data, and frailty analyses (AIM 2) included those with frailty data. Results/Anticipated Results: We anticipate that INSTI use will be associated with a higher prevalence of sarcopenia, pre-frailty, and frailty compared to PLWH on non-INSTI ART and persons without HIV (PWOH). Specifically, we expect greater declines in muscle strength, grip strength/BMI, gait speed, and overall physical function in the INSTI group. Furthermore, we predict that the INSTI group will show increased frailty markers, such as unintentional weight loss, weakness, and slow walking speed, over time. These findings could highlight a critical need for monitoring physical health in PLWH on INSTIs. Discussion/Significance of Impact: PLWH may experience sarcopenia and frailty earlier than those without HIV, and INSTIs might contribute. Given their widespread use, it is essential to assess the link to improve prevention and care strategies for those affected.