The utilization of storage lipids and their associated fatty acids (FA) is an important means for organisms to cope with periods of food shortage, however, little is known about the dynamics and FA mobilization in benthic copepods (order Harpacticoida). Furthermore, lipid depletion and FA mobilization may depend on the ambient temperature. Therefore, we subjected the temperate copepod Platychelipus littoralis to several intervals (3, 6 and 14 days) of food deprivation, under two temperatures in the range of the normal habitat temperature (4, 15 °C) and under an elevated temperature (24 °C), and studied the changes in FA composition of storage and membrane lipids. Although bulk depletion of storage FA occurred after a few days of food deprivation under 4 °C and 15 °C, copepod survival remained high during the experiment, suggesting the catabolization of other energy sources. Ambient temperature affected both the degree of FA depletion and the FA mobilization. In particular, storage FA were more exhausted and FA mobilization was more selective under 15 °C compared with 4 °C. In contrast, depletion of storage FA was limited under an elevated temperature, potentially due to a switch to partial anaerobiosis. Food deprivation induced selective DHA retention in the copepod's membrane, under all temperatures. However, prolonged exposure to heat and nutritional stress eventually depleted DHA in the membranes, and potentially induced high copepod mortality. Storage lipids clearly played an important role in the short-term response of the copepod P. littoralis to food deprivation. However, under elevated temperature, the use of storage FA as an energy source is compromised.