Background <p>Egg-derived phosphatidylcholine may support the maintenance of cognitive function. However, its metabolite trimethylamine N-oxide is considered a potential cardiovascular risk factor. The present study aimed to assess, for the first time, the effects of long-term intake of egg-derived phosphatidylcholine on serum trimethylamine N-oxide concentrations in healthy Japanese middle-aged and older adults.</p> Methods <p>A secondary analysis was conducted using preserved serum samples obtained from a previous randomized controlled trial (UMIN000041554). The study population, comprising thirty-two healthy middle-aged and older Japanese adults, was categorized into four groups based on their level of phosphatidylcholine intake: placebo (0&#xa0;g/day), low-dose (0.3&#xa0;g/day), medium-dose (1.0&#xa0;g/day), and high-dose (3.0&#xa0;g/day). Serum trimethylamine N-oxide concentrations were measured before and after the 12-week intervention.</p> Results <p>In the high-dose phosphatidylcholine group, serum trimethylamine N-oxide concentration increased significantly at 12&#xa0;weeks (Week 12) compared with Baseline (Week 0) (<i>P</i> = 0.028). In contrast, no significant between-group differences were observed in serum trimethylamine N-oxide concentrations or in the magnitude of change from Baseline to Week 12, including comparisons with placebo. Across all groups, mean trimethylamine N-oxide values remained substantially below the threshold associated with high clinical risk.</p> Conclusions <p>Twelve weeks of egg-derived phosphatidylcholine supplementation did not result in serum trimethylamine N-oxide concentrations approaching clinically concerning levels under the specific conditions tested. These findings suggest that egg-derived phosphatidylcholine may serve as a dietary source of choline without inducing a substantial elevation in trimethylamine N-oxide in healthy middle-aged and older adults. Confirmation of these results in studies with larger sample sizes is warranted.</p>