Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. Identification of these factors is facilitated by high-throughput analyses of behavior in zebrafish larvae, which can be imaged in multiwell or multilane plates. However, the nutritional needs of zebrafish larvae during the behavioral experiments are not fully understood. Zebrafish larvae begin feeding between 4 and 5 days postfertilization (dpf), but can live solely on nutrients derived from the yolk until at least 7 dpf. To examine whether feeding affects behavior, we measured a broad range of behaviors with and without feeding at 5, 6, and 7 dpf. We found that feeding did not have a significant effect on behavior in 5-day-old larvae. In contrast, fed 6- and 7-day-old larvae displayed increased avoidance responses to visual stimuli, increased swim speeds, and decreased resting in comparison to unfed larvae. In addition, the fed 7-day-old larvae displayed a decrease in thigmotaxis and a decrease in the distance between larvae in the presence of visual stimuli. Thus, feeding affects a range of behaviors in 6- and 7-day-old larvae. We conclude that 5-day-old larvae are well-suited for high-throughput analyses of behavior, since effects of feeding can be avoided at this time. For high-throughput analyses of behavior in older larvae, standard feeding protocols need to be developed.