The interior holds five successive layers of painting, applied and overpainted across the early Christian era, the 9th century, and the 10th, 11th and 13th. During restoration, some layers were carefully detached and repositioned once the earliest was removed, so frescoes from wildly different periods now share the same space. The oldest, from the 7th century, shows apostles ringing the bottom of the sanctuary conch, with Saint Isidore beside a window. Over them, in the 9th-century Iconoclast period, painters added only crosses and ornament, no human figures, obeying an empire that had forbidden sacred images. Each layer is a frozen moment of changing faith.

The building itself is older than its paintings. It began life as an early Christian basilica, and from that first structure only the synthronon, the tiered bench for clergy, and the bishop's throne in the curved sanctuary still survive. In middle Byzantine times the basilica was rebuilt as a transitional cross-in-square church, the compact domed form that would define Byzantine architecture. Later a domed narthex was added at the entrance, with a small chapel dedicated to Saint Akindynos on one side and a vaulted room on the other, the church growing in stages across hundreds of years.

An inscription carved on a stone beam, probably once part of the templon screen, records a renovation in 1052 and names the people who paid for it: Bishop Leo, a man called Niketas who bore the imperial titles protospatharios and tourmarches of Naxia, and Count Stephen Kamilaris. A second inscription in the north chapel commemorates the passing of a woman named Anna in 1056. These are not famous names from the chronicles. They are ordinary, devout islanders, fixed in stone nearly a thousand years ago, their commission still standing exactly where they meant it to.

What makes Protothronos remarkable is not only its age but its continuity. While other ancient churches became ruins or museums, this one has held services without interruption since the 11th century, making it one of the oldest continuously functioning churches in Greece. Later inscriptions from the 16th and 17th centuries record still more repairs, the steady upkeep of a building people refused to abandon. To attend a service here is to join a thread of worship unbroken for nearly a thousand years, beneath the gaze of saints painted before the Crusades.