(a) Three-month-old male mice were split into two groups and assigned to ad libitum (AL) or dietary restricted (DR) food supply (animals were matched by body mass and food intake). DR animals were offered 60% of AL intake as one food ration per day. After 9 months of diet (at the age of 12 months), mice were split into 4 groups liver-weight to body-weight: AL (remaining on AL feeding, n=9), DR (remaining on DR feeding, n=10), AL to DR (switching from AL to restricted food supply, n=7), and DR to AL (switching from restricted to AL food supply, n=7). Animals stayed on the assigned food regime for 3 months until killed at 15 months of age. (b) Liver-weight to body-weight ratios in all the experimental groups. Data per animal (dots) and means±s.d. are shown. (c) Micrographs showing Oil Red O staining on frozen liver sections in 15-month-old animals in the indicated groups (red=Oil Red O, blue=haematoxylin, scale bar 100 μm). (d) Percentage of Oil Red O staining was determined using ImageJ (n=5). (e) Representative micrographs showing decreased Nile red staining in DR in comparison to AL animals at 15 month of age (scale bar 20 μm, in Merge: blue=DAPI, red=Nile red, green=Actin green). Grade of steatosis was independently assessed by a liver pathologist who confirmed Oil Red O and Nile red results. All data are mean±s.d. with 5–10 animals per group. Significant differences (one-way analysis of variance) are indicated with *P≤0.05 and **P≤0.001.