Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60mg/kg) induced diabetic ApoE-KO mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 weeks and during cycles 1 and 5 caloric measurements were performed. After 10 weeks, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 (HPSE-1) activity, and spatial mass spectrometry imaging (MSI) to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increased fatty acid oxidation. Overall bodyweight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary HPSE-1 activity. Weight loss was found to be of lean mass and water, not of fat mass. While capillary loop morphology and endothelial glycocalyx heparan sulfate contents was preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. MSI further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different then diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.