Identification of low-grade lameness is challenging. A whole horse ridden ethogram has been developed, describing 24 behavioural markers. Previous work indicated that the presence of ≥8 behavioural markers was likely to reflect musculoskeletal pain. The objectives of this repeated measures study were to compare the results of application of the ridden-horse ethogram by trained and untrained assessors to horses before and after musculoskeletal pain had been substantially improved using diagnostic analgesia, and to assess the repeatability of the ethogram application among untrained assessors, and to compare their performance with a trained assessor. All horses underwent a comprehensive lameness investigation. Anonymised video recordings of 21 lame horses, ridden by professional riders in trot and canter before and after diagnostic analgesia had abolished lameness, were reviewed in a random order by a trained assessor and 10 untrained assessors. For each horse the duration of the recordings before and after diagnostic analgesia was time matched. The most frequent lameness grade was 2/8 (range 1-4). For the trained assessor, the number of behaviours exhibited by lame horses before diagnostic analgesia ranged from 3-12/24 (median 10; mean 8.9). After lameness and overall performance had been substantially improved using diagnostic analgesia, the number of behaviours ranged from 0-6/24 (median 3; mean 3.0). The decrease in behaviour scores for all assessors after diagnostic analgesia was highly significant (Z = 20,147, P<0.0001). Agreement between the trained assessor and untrained assessors was moderate before diagnostic analgesia and nonexistent after analgesia (Fleiss Kappa 0.49, 0 respectively), when individual behaviours were assessed. The main limitation was that horses were anonymised, but it was impossible to blind their identity, so bias is possible. It was concluded that despite limitations in the agreement between untrained observers and the trained assessor, the ethogram is a potentially valuable tool for determining the presence of musculoskeletal pain and may be useful for longitudinal monitoring of improvement in lameness.