The impact of fasting on IHL (intrahepatic lipid) content in human subjects has not been investigated previously, but results indicate that it may change rapidly in response to metabolic cues. The aim of the present study was to measure IHL content after fasting and to correlate this with circulating lipid intermediates. A total of eight healthy non-obese young males were studied before and after 12 or 36 h of fasting. IHL content was assessed by ¹H-magnetic resonance spectroscopy, and blood samples were drawn after the fasting period. IHL content increased significantly after the 36 h fasting period [median increase 156% (range, 4–252%); P<0.05]. Furthermore, a significant positive correlation between this increase and 3-hydroxybutyrate concentration was detected (P=0.03). No significant change in IHL content was demonstrated after the 12 h fasting period. The baseline median inter-individual variation in IHLs was 0.51% (range, 0.25–0.72%). The coefficient of variation of IHL measurements was 11.6%; 25–30% of the variation was of analytical origin and the remaining 70–75% was attributed to repositioning. In conclusion, IHL content increases in healthy male subjects during fasting, which demonstrates that nutritional status should be accounted for when assessing IHLs in clinical studies. Moreover, the increase in IHLs was positively correlated with the concentration of 3-hydroxybutyrate.