Marine phytoplankton can evolve rapidly when confronted with aspects of climate change because of their large population sizes and fast generation times. Despite this, the importance of environment fluctuations, a key feature of climate change, has received little attention—selection experiments with marine phytoplankton are usually carried out in stable environments and use single or few representatives of a species, genus or functional group. Here we investigate whether and by how much environmental fluctuations contribute to changes in ecologically important phytoplankton traits such as C:N ratios and cell size, and test the variability of changes in these traits within the globally distributed species Ostreococcus. We have evolved 16 physiologically distinct lineages of Ostreococcus at stable high CO<inf>2</inf> (1031±87 μatm CO<inf>2</inf>, SH) and fluctuating high CO<inf>2</inf> (1012±244 μatm CO<inf>2</inf>, FH) for 400 generations. We find that although both fluctuation and high CO<inf>2</inf> drive evolution, FH-evolved lineages are smaller, have reduced C:N ratios and respond more strongly to further increases in CO<inf>2</inf> than do SH-evolved lineages. This indicates that environmental fluctuations are an important factor to consider when predicting how the characteristics of future phytoplankton populations will have an impact on biogeochemical cycles and higher trophic levels in marine food webs.