This study assessed the composition of digesta from captive and wild Acipenser fulvescens to consider how food availability and dietary absorption may influence stocking survival. Non-lethal gastric lavage and colonic flushing were evaluated in laboratory fish and then in wild A. fulvescens caught from the Tennessee River. Gastrointestinal contents were collected at 2, 4, 6, 12, 24, and 32 h post-feeding. The stomach content relative to ingested food was 36.67% ± 5.09% at 2 h post-feeding, peaking at 4 h (75.82% ± 14.09%), and no food was recovered after 12 h. The colonic solid waste (g/kg) increased with time, with the highest recovery (5.40 ± 1.30 g/kg) at 32 h. Proximate composition of collected samples was determined using standard colorimetric assay protocols. There was a significant reduction in protein concentration in the stomach starting at 2 h, and glucose concentrations were stable in both stomach and colonic contents from 2 to 32 h post-feeding in laboratory animals, suggesting longer processing times for dietary glucose compared to other nutrients. The protein concentration of recovered stomach solids was very similar between laboratory and wild populations (46.17 ± 7.32 vs. 40.00 ± 6.57 mg/dL) and in colonic solids (22.99 ± 4.26 vs. 19.86 ± 3.95 mg/dL). Lipid concentrations, however, deviated substantially, with 40.11 ± 7.71 mg/dL in stomach and 35.59 ± 7.29 mg/dL in faeces of laboratory fish, and only 18.26 ± 3.47 mg/dL (stomach) and 22.00 ± 5.54 mg/dL (colonic) in wild fish.Glucose represented a relatively small fraction in both stomach and colonic contents and differed based on habitat only in the colon, with wild fish retaining complex sugars for longer periods. These results indicate that high-energy diet components are either reduced in natural diets or are preferentially absorbed within the first 12 h compared to laboratory fish. More study on use of lipid-dense foods in the riverine environment may provide information to improve the survival of A. fulvescens to support their reintroduction.