The size of your flies could affect their behavior, but it may depend on the type of behavior you are measuring.
Fly size can be directly proportional to the density of flies in the parental vial. Really crowded vials produce smaller flies and sparsely populated vials produce large flies. This is important when considering behavior because it can reflect the nutritional condition of the fly (few food resources during development results in smaller flies). You can imagine if you are studying some type of behavior that involves food in some way (feeding, taste response, foraging, appetitive memory, and any type of motivational response involving food deprivation) that nutritional rearing conditions might impact this.
Fly size can also impact social dynamics. Size of males and females can affect courtship (Partridge et al, 1987), immediate mating success and competitive fertilization (De Nardo et al, 2021), lifetime mating success (Partridge & Farquhar, 1983), copulation speed and success (Jagadeeshan et al, 2015), and how much receptivity-inhibiting sex peptide is transferred in ejaculate (Wigby et al, 2016).
I also suspect that size affects aggression. While I couldn’t find any reports of this happening in Drosophila, there is a social hierarchy that exists resulting in winners and losers during fights over resources like food and mates in Drosophila (Yurkovic et al, 2006; Trannoy et al, 2016). In other species, these types of hierarchies are affected by body size.
Body size is also important for basic locomotor responses since large and small flies can move at different speeds, with different angles. It can also affect the number of tracking errors you get. We find that if the flies are too small we tend to get more tracking errors in the software that we have used in the lab.
So how can you make sure your flies have consistent body size?
Make sure the density of your parental vials is consistent (not too dense and not too sparse)
Make sure your control and experimental flies are reared at the same temperature (warmer temperatures like 30°C can produce smaller flies than lower temperatures like 18°C)
Use high quality food (more on this at ‘Which fly food recipe should I use to raise flies for behavior?’)
Make sure the quality of the food is consistent (that is not too wet, not too dry, and doesn’t have slime or strange colors (see pictures at the bottom of ‘Which fly food recipe should I use to raise flies for behavior?’)
Make sure your flies are the same age
Allow the parents to only lay eggs for 1-2 days, that way you’ll have (mostly) age-matched progeny.
If you are using vials in which the parents were laying eggs for many days, make sure your flies are all from the same eclosion wave. By this, I mean if you are using a vial and taking a number of collections from the same vial, the flies that eclose first will typically be a bit bigger than the flies that eclose the next day, or the next day after that. We typically collect from one or two waves of eclosion because you will get too much variation in size if you collect from the 3rd or 4th wave of eclosion from slightly older vials.
Sometimes you just get variation in fly size. A good rule of thumb is to look for size differences of your flies when you are collecting them. If you suspect that they might be different sizes, take pictures of a few example collections and make a note in your lab notebook.