Purpose <p>In patients undergoing hemodialysis, poor nutritional status frequently occurs due to depletion of essential nutrients, amino acids, during dialysis sessions. Protein supplementation during dialysis is becoming common to counteract this loss. We compared effects of intradialytic ingestion of fish protein, a novel protein source, with those of the traditionally used whey protein, on blood amino acid levels in patients undergoing hemodialysis.</p> Methods <p>Ten outpatients aged ≥ 60&#xa0;years undergoing hemodialysis were enrolled in this single-intervention crossover study. Participants underwent three mid-week dialysis sessions with a one-week washout period. During the first session, blood and spent dialysate samples were collected without protein ingestion. In subsequent sessions, participants consumed fish or whey protein soups 30&#xa0;min after dialysis initiation, followed by blood and spent dialysate sample collection. The effects on amino acid concentrations, including those of branched-chain amino acids (BCAAs), were analyzed using a mixed model for repeated measures.</p> Results <p>Both proteins significantly elevated BCAA concentrations compared with usual care. The effect of fish protein persisted until the end of dialysis (p = 0.016), whereas that of whey protein lasted up to 180&#xa0;min after dialysis initiation (p = 0.002). Whey protein intake resulted in significantly higher amino acid levels than fish protein intake 30–90&#xa0;min post-ingestion (p &lt; 0.001), with no differences observed thereafter.</p> Conclusion <p>Both fish and whey protein intake increased amino acid levels compared with usual care. Whey protein induced a rapid increase in levels, whereas fish protein included a slower but sustained increase in levels.</p> Trial registration number and date of registration <p>The study protocol was registered in the UMIN Clinical Trials Registry (UMIN000050805).</p>