Habitat loss and fragmentation are among the biggest threats to tropical biodiversity and associated ecosystem services. We examined forest dynamics in a mid-elevation 365-ha fragment in southern Costa Rica. The fragment was isolated in the mid-1970s and belongs to the Las Cruces Biological Station. A 2.25-ha permanent plot was established in the center of the old-growth forest (>400 m to nearest edge boundary) and all plants >5 cm DBH were censused, mapped, and identified to species in two surveys taken ~5-6 years apart (>3,000 stems/survey). Although the reserve maintains high species richness (>200 spp.), with many rare species represented by only one individual, we document a strong shift in composition with a two-fold increase in the number of soft-wooded pioneer individuals. The dominant late-successional understory tree species, Chrysochlamys glauca (Clusiaceae), and most species in the Lauraceae, declined dramatically. Turnover was high: 22.9% of stems in the first survey were lost, and 27.8% of stems in the second survey represented new recruits. Mean tree diameter decreased significantly and there was a 10% decrease in overall biomass. Such alteration has been documented previously but only in smaller fragments or within ~100 m of an edge boundary. Further penetration into this fragment was perhaps driven by a progressive invasion of disturbance-adapted species into the fragment's core over time; the loss of once-dominant late successional species could be a contributing factor. The pattern found is of particular concern given that such fragments represent a substantial portion of today's remaining tropical habitat; further studies in similar-sized fragments that have been isolated for similar prolonged periods are called for.