Fibroblast growth factor 21 (FGF21) agonists have shown promising effects in preclinical models of non-alcoholic fatty liver disease (NAFLD) as well as in short-term clinical trials in patients with non-alcoholic steatohepatitis (NASH). Comparing drug formulation, dose, administration route and age, this exploratory study investigated effects of FGF21 on NAFLD-associated measures in a validated guinea pig model. In three separate studies, female guinea pigs received a high-fat diet prior to intervention with escalating doses of either recombinant native human FGF21 or a human FGF21 human recombinant analogue (FGF21/19 chimer) with an extended half-life. While no significant effects of native FGF21 on the investigated endpoints were observed, the long-acting FGF21/19 chimer significantly altered the levels of circulating lipids, increasing plasma concentrations of cholesterol (TC, LDLc and HDLc) in young guinea pigs (p < 0.01 for all three parameters). Relative liver weights were reduced in FGF21/19-treated young animals (p < 0.05) compared to mature animals, whereas FGF21/19 reduced body weights in both age groups (p < 0.001). The FGF21/19 chimer effects on dyslipidemia, body and liver weights particularly in young animals, support an age-associated difference in the FGF21 response. The limited effects of the native human FGF21 highlights potential species-associated differences of this compound.