Splitting apheresis platelet (PLT) units increase available inventory during shortages. The impact of prolonged storage in gas-impermeable aliquot bags on PLT quality in vitro and transfusion outcomes in patients remains uncertain. We assessed in vitro PLT quality and thromboelastography (TEG) in PLTs stored for 8 or 24 h in aliquot bags compared with baseline (T0). Retrospective assessment of response (PLT increment and corrected count increment (CCI)) was conducted among adults (≥18 years) transfused with split platelet units from January 2021 to June 2022. No differences were observed in PLT and white blood cell (WBC) counts, mean platelet volume, or TEG parameters during storage, except for an increase in TEG R time (mean ± SD) at 24 h (6.1 ± 0.5 min) compared to T0 (4.4 ± 0.8 min), p = 0.0031 one-way ANOVA. Eighty-one patients were transfused 119 split units with a median [IQR] PLT yield of 2.1 × 1011[1.9 × 1011 to 2.3 × 1011] and storage duration of 1.6[0.7-9.1] h. The overall median PLT count increment was 6.0 × 103/uL and CCI was 5.0 × 103, correlating negatively with split unit storage duration (Spearman rho = -0.218, p = 0.017). Compared with split transfusions of pathogen-reduced (PR) PLTs, non-PR splits were associated with higher median platelet count increments (7.0 × 103/μL vs. 4.0 × 103/μL, p = 0.0263 Mann-Whitney U) and higher CCIs (6.5 × 103 vs. 3.9 × 103, p = 0.0116 Mann-Whitney U) despite no differences in PLT yields (2.1 × 1011/μL vs. 2.1 × 1011/μL). Storing PLTs in aliquot bags for 8 or 24 h does not adversely affect their quality in vitro. Splitting apheresis PLTs are feasible for adult transfusions during shortages. It may be advisable to prioritize non-PR PLTs for splitting given improved patient responses.