Fibroblast growth factor-21 (FGF-21), is a protein involved in cell growth and differentiation, development, wound repair and metabolism. Research looking at the impact of weight loss on FGF-21 levels is limited. The objective of this exploratory study was to determine changes in serum FGF-21 levels following weight loss induced by either continuous energy restriction or intermittent energy restriction. A sub cohort of participants who completed a 12-month dietary intervention trial following continuous energy restriction, or a week-on week-off energy restriction pattern, were selected for analysis. FGF-21 levels were not altered by weight loss and were not correlated with body weight or BMI at baseline or 12 months. Weight loss after 12 months either through continuous energy restriction or intermittent energy restriction was −5.9 ± 4.5 and −4.9 ± 3.4 kg, respectively. There was no change in FGF-21 levels, 0.3 ± 0.9 and 0.04 ± 0.2 ng/mL (p = 0.2). In conclusion, weight loss in healthy overweight or obesity subjects did not affect FGF-21 levels.