We developed a new technique to observe macroautophagy in the brain in vivo, and examined whether fasting induced macroautophagy in neurons and how the induction was different between Alzheimer's disease (AD) model and control mice. Lentivirus for EGFP-LC3 injected into the brain successfully visualized autophagosome in living neurons by two-photon microscopy. The time-lapse imaging revealed that fasting increased the number, size and signal intensity of autophagosome in neurons. In AD model mice, these parameters of autophagosome were higher at the basal levels before starvation, and increased more rapidly by fasting than in control mice. However, metabolism of exogenous labeled Aβ evaluated by the new technique suggested that the activated macroautophagy was insufficient to degrade the intracellular Aβ increased by enhanced uptake from extracellular space after fasting. Ordinary immunohistochemistry also revealed that fasting increased intracellular accumulation of endogenous Aβ, triggered cell dysfunction but did not mostly decrease extracellular Aβ accumulation. Moreover, we unexpectedly discovered a circadian rhythm of basal level of macroautophagy. These results revealed new aspects of neuronal autophagy in normal/AD states and indicated usefulness of our method for evaluating autophagy functions in vivo.