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This prospective observational trial investigated effects and safety of periodic fasting in subjects with and without type 2 diabetes mellitus (T2DM). The primary end point was set as the change of fatty liver index (FLI) as a surrogate parameter of non-alcoholic fatty liver disease (NAFLD). Six-hundred and ninety-seven subjects (38 with T2DM) were enrolled. A baseline FLI ≥ 60 (the threshold for fatty liver) was found in 264 subjects (37.9%). The mean duration of fasting was 8.5 ± 4.0 days (range 6–38). FLI decreased significantly (−14.02 ± 11.67; <i>p</i> &lt; 0.0001), with a larger effect in individuals with T2DM (−19.15 ± 11.0; <i>p</i> &lt; 0.0001; <i>p</i> = 0.002 compared to non-diabetic subjects). Body mass index (BMI) decreased by −1.51 ± 0.82 kg/m², and 49.9% of the subjects lost ≥5% body weight. After fasting, nearly half of the 264 subjects with FLI ≥ 60 (highest risk category) shifted to a lower category. The improvement of FLI correlated with the number of fasting days (r = −0.20, <i>p</i> &lt; 0.0001) and with the magnitude of BMI reduction (r = 0.14, <i>p</i> = 0.0001). Periodic fasting with concomitant weight reduction leads to significant rapid improvement of FLI in subjects with and without T2DM.