The aim of this study is to validate whether OCFAs (Odd chain fatty acids), specifically, pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) are potential biomarkers of fermentable fiber in two independent studies using a validated analytical method by Zhao et al (Zhao et al., 2017). OCFAs were first assessed in a fiber supplementation study in which 21 participants received 10g/day dietary fiber for 7 days, with blood samples collected on the final day at a 420-minute study visit. Details regarding participant recruitment and eligibility, demographics, dietary interventions, study design have been published elsewhere (Byrne et al., 2019). OCFAs were further assessed in a highly controlled inpatient setting in which 19 participants consumed a high fiber (45.1g/day) and a low fiber diet (13.6g/day) for 4 days. Details regarding participant recruitment and eligibility, demographics, dietary interventions and study design have been published elsewhere (Garcia Perez et al., 2017). Sample from both studies were prepared following the extraction and derivatization protocol previously developed by Zhao et al (Zhao et al., 2017). Raw data from GC-MS targeted analysis were exported to MassHunter Qualitative Analysis Software (vB.07.01). The targeted metabolites were identified by comparing mass spectra and retention index in the in-house reference library. Thereafter, the dataset was exported to MassHunter Quantitative Analysis Software (vB.07.0) to perform baseline correction, smoothing, noise reduction, deconvolution, library searching and data integration in each chromatogram from samples and the calibration standards. The responses of the sample and standards were normalized by calculating the ratio of the peak area and the corresponding area of the internal standard as the corrected area. The squared correlation coefficient (R2) was used as a measure of linearity of the calibration curve in which the known amounts of C15:0 and C17:0 was linearly regressed against corrected area. In the first study we found that there was no effect of fiber supplementations on the serum concentrations of C15:0 and C17:0, as the changes in OCFAs concentrations were not different between control and dietary fibre supplementations (C15:0, p=0.404; C17:0, p=0.288). There were no differences in the levels of OCFAs over time, as the levels of OCFAs were not different between 0, 240, 420 minutes (C15:0, p=0.074; C17:0, p=0.503). In the second study, no differences in fasting OCFAs concentrations were found between a high fiber (45.1g/day) and a low fiber diet (13.6g/day) after 4-day inpatient period. Furthermore, OCFAs levels were not significantly different between high and low fiber diets measured after breakfast and after lunch (C15:0, p=0.286, p=0.586; C17:0, p=0.744, p=0.433 respectively). Therefore, current work do not support the proposed relationship between dietary fiber and OCFAs and suggests that further validation is required.