BDNF signalling in hypothalamic neuronal circuits is thought to regulate mammalian food intake. In light of this, we investigated how a lifestyle intervention influenced serum levels and DNA methylation of BDNF gene in fat tissue and buffy coat of NDH individuals. In total, 20 participants underwent anthropometric measurements/fasting blood tests and adipose tissue biopsy pre-/post-lifestyle (6 months) intervention. DNA was extracted from adipose tissue and buffy coat, bisulphite converted, and pyrosequencing was used to determine methylation levels in exon IV of the BDNF gene. RNA was extracted from buffy coat for gene expression analysis and serum BDNF levels were measured by ELISA. No differences were found in BDNF serum levels, but buffy coat mean BDNF gene methylation decreased post-intervention. There were correlations between BDNF serum levels and/or methylation and cardiometabolic markers. (i) Pre-intervention: for BDNF methylation, we found positive correlations between mean methylation in fat tissue and waist-hip ratio, and negative correlations between mean methylation in buffy coat and weight. (ii) Post-intervention: we found correlations between BDNF mean methylation in buffy coat and HbA1c, BDNF methylation in buffy coat and circulating IGFBP-2, and BDNF serum and insulin. Higher BDNF % methylation levels are known to reduce BNDF expression. The fall in buffy coat mean BDNF methylation plus the association between lower BDNF methylation (so potentially higher BDNF) and higher HbA1c and serum IGFBP-2 (as a marker of insulin sensitivity) and between lower serum BDNF and higher circulating insulin are evidence for the degree of BDNF gene methylation being implicated in insulinisation and glucose homeostasis, particularly after lifestyle change in NDH individuals.