The aim of this study was to examine sex- and age-dependent changes in carbohydrate and lipid reserves throughout the fourth larval instar of Chironomus riparius Meigen. Individuals were sexed using genital discs and aged using phases of development defined through the observation of genital and thoracic imaginal discs. Glycogen concentration was measured in the eviscerated body (fat body, muscles, and cuticle), and the levels of glucose and trehalose were followed in the hemolymph, whereas the amounts of free glycerol and triglycerides were measured in both tissues. Compared with males, females had higher levels of body reserves, whereas the levels of sugars in the hemolymph were similar for both sexes, except for free glucose, which was slightly higher in males. The levels of energy-yielding substrates in both the eviscerated body and hemolymph varied significantly with the phase of development for both sexes, except for the concentration in free glycerol in the hemolymph. Analysis of variability indicated that selection of larvae for both the sex and phase of development significantly reduces uncertainty of biochemical measures. The results are discussed with respect to potential use of the levels of energy-yielding substrates as endpoints in toxicity tests with fourth-instar C. riparius larvae.