Freshwater ecosystems, especially springs, are highly sensitive to environmental changes. They are also excellent natural laboratories because of their stable conditions, reducing the number of variables to be considered in field studies. We examined the composition, dynamics and colonisation patterns of macroinvertebrates with respect to canopy coverage and time of day in which available areas are actively colonised. We used artificial substrates that mimicked the natural habitat structure at an isolated karst spring and recovered exposed substrates every 12 h. Physico-chemical parameters of water did not differ significantly regardless of canopy cover. The most numerous representatives and the pioneering champions were larvae of Baetidae (Ephemeroptera) and Chironomidae (Diptera). Simuliidae were also among the most successful pioneering species. Most observed groups more actively colonised substrates in the closed canopy area. Oligochaeta and Gammarus fossarum were more numerous on substrates in the open canopy area. Individuals of all analysed groups showed day-night migration patterns and were more active at night. Coleoptera ( Elmis sp.) were the poorest (re)colonisers among the analysed taxa.