The trophic structure of Ascension Island's sub-tidal reef assemblages is poorly understood. Unlike other tropical reef systems, sub-tidal habitats have very low abundance of both coral and macrophyte species. Visually dominant is a diverse assemblage of fish species, with particularly high densities of Melichthys niger, a voracious omnivore. In contrast, the nocturnal species assemblage is notably different, visually dominated by benthic invertebrates. To quantify the difference between day and night visible assemblages, we conducted day/night pairs of transect surveys of fish and invertebrates across three depths, and spanning 9 months, assigning all species to one of 10 functional groups. Multivariate analysis of surveys revealed significant turnover in species between day and night surveys and between survey periods, with concomitant changes in species rank-abundance distributions. Juveniles of a number of fish species were determinate in observed differences. Conversely, diversity of functional groups between day/night surveys and between seasons were not different, however there was significant species turnover within functional groups between day and night assemblages. The lack of proportional change in functional groups but a turn-over of species between day and night assemblages suggest that there may be a degree of functional redundancy in Ascension Island's marine trophic profile. Further investigation into the spatio-temporal variation in trophic profile and functional diversity around the island will benefit conservation and fisheries management in this isolated and poorly understood marine system.