Higher dietary protein intake is increasingly recommended for the elderly; however, high protein diets have also been linked to increased cardiovascular disease (CVD) risk. Trimethylamine-N-oxide (TMAO) is a bacterial metabolite derived from choline and carnitine abundant from animal protein-rich foods. TMAO may be a novel biomarker for heightened CVD risk. The purpose of this study was to assess the impact of a high protein diet on TMAO. Healthy men (74.2 ± 3.6 years, n = 29) were randomised to consume the recommended dietary allowance of protein (RDA: 0.8 g protein/kg bodyweight/day) or twice the RDA (2RDA) as part of a supplied diet for 10 weeks. Fasting blood samples were collected pre- and post-intervention for measurement of TMAO, blood lipids, glucose tolerance, insulin sensitivity, and inflammatory biomarkers. An oral glucose tolerance test was also performed. In comparison with RDA, the 2RDA diet increased circulatory TMAO (p = 0.002) but unexpectedly decreased renal excretion of TMAO (p = 0.003). LDL cholesterol was increased in 2RDA compared to RDA (p = 0.049), but no differences in other biomarkers of CVD risk and insulin sensitivity were evident between groups. In conclusion, circulatory TMAO is responsive to changes in dietary protein intake in older healthy males.