Prior investigation has suggested a positive association between increased colonic propionate production and circulating odd-chain fatty acids [(OCFAs; pentadecanoic acid (C15:0), heptadecanoic acid (C17:0)]. As the major source of propionate in humans is the microbial fermentation of dietary fiber, OCFAs have been proposed as candidate biomarkers of dietary fiber. The objective of this study is to critically assess the plausibility, robustness, reliability, dose-response, time-response aspects of OCFAs as potential biomarkers of fermentable fibers in two independent studies using a validated analytical method. OCFAs were first assessed in a fiber supplementation study, where 21 participants received 10g dietary fiber supplementation for 7 days with blood samples collected on the final day at a 420 minute study visit. OCFAs were then assessed in a highly controlled inpatient setting, which 19 participants consumed a high fiber (45.1g/day) and a low fiber diet (13.6g/day) for 4 days. Collectively in both studies, dietary intakes of fiber as fiber supplementations or having consumed a high fiber diet did not increase circulating levels of OCFAs. The dose and temporal relations were not observed. Current study has generated new insight on the utility of OCFAs as fiber biomarkers and highlighted the importance of critical assessment of candidate dietary biomarkers before application.