Circadian rhythms are ubiquitous in eukaryotes, and co-ordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants1,2. This daily timekeeping is thought to be driven by transcriptional/translational feedback loops, whereby rhythmic expression of clock gene products regulates expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms3. The unicellular pico-eukaryotic alga, Ostreococcus tauri, possesses a naturally minimised clock, which includes many features that are shared with higher eukaryotes (plants), such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes4. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping5, a reappraisal of the transcriptional contribution to oscillator function is overdue. Here we show that non-transcriptional mechanisms are sufficient to sustain circadian timekeeping in the eukaryotic lineage, though they normally function in conjunction with transcriptional components. We identify oxidation of peroxiredoxin proteins as a transcription-independent rhythmic biomarker, which is also rhythmic in mammals6. Moreover we show that pharmacological modulators of the mammalian clockwork have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, appear to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria7, and are conserved across kingdoms.