Purpose: End-stage anteromedial knee OA (AMOA) is associated with limited lateral joint space narrowing osteophytosis or synovitis. Synovial fluid (SF) is a non-Newtonian biological fluid with a complex non-homogenous matrix. Despite the free flow of SF within the knee, this raises the possibility of compartment-specific SF biology. Equilibrium in SF composition between knee joint compartments is assumed, but has never been formally tested. Anatomical variations in SF analysis would have important practical implications for the consistency of sampling needed for biomarker research, especially for unicompartmental OA. This study aims to determine if the anatomical site of sampling affects SF analysis in patients with end-stage AMOA and tri-compartmental OA (TCOA). Methods: SF was collected from the medial, lateral and patello-femoral (PF) compartments of patients with AMOA (n=8) and TCOA (n=8) at the time of primary arthroplasty (total N=16). Site-specific SF collection (∼250μl/compartment) was done simultaneously prior to arthrotomy. Samples underwent identical processing, storage and preparation. Samples were analysed for 9 pro-inflammatory cytokines (IL1β, TNFα, IL6, IL8, IL15, IL2, IL12, IL17 & GMCSF), 4 regulatory cytokines (IL1RA, IL4, IL10, IL2R), 7 chemokines (Eotaxin, IP10, MCP1, MIG, MIP1α, MIP1β, RANTES) and TGFβ1, -β2 & -β3. Each of the three joint compartments was given a Kellgren & Lawrence grade (KLG), and the OARSI system was applied to the medial and lateral compartments only as originally described. Within-subject comparisons of KLG and OARSI scores for joint space narrowing (JSN) and osteophytosis (OP) between joint compartments were conducted separately for patients with AMOA and TCOA. P<0.05 was considered statistically significant. The agreement of SF measurements between compartments was assessed for each analyte using the coefficient of variation (CV) and intraclass correlation coefficient (ICC). The acceptable threshold for cross-compartment CV (cCV) was set at 20%, which is the threshold for acceptable intra- and inter-assay precision. An ICC >0.80 was considered excellent absolute agreement. Results: Compartment KLG (p<0.001), OARSI JNS (p=0.012) and OARSI OP (p=0.030) scores for AMOA were significantly higher in the medial compartment, but there was no significant difference between sites for TCOA. In both AMOA and TCOA patients, only 10 analytes were quantifiable in ≥75% (6/8) of compartment-matched samples to allow meaningful analysis: 4 pro-inflammatory cytokines (IL6, IL8, IL12 & IL15); 3 chemokines (Eotaxin, MCP1 & IP10); and the 3 isoforms of TGFβ. The median cCV for all analytes was <15% in both AMOA and TCOA patient groups. The median cCV was significantly less than 20% (one sample Wilcoxon signed rank test) for all analytes in both patient groups. The ICC for absolute agreement was >0.80 for all analytes in the both patient groups. Seven (out of 10) analytes had ICCs >0.90 in both groups. Conclusions: There is excellent agreement between compartment-specific SF measurements in both AMOA and TCOA. The anatomical site of sampling does not affect SF analysis even when there are significant compartmental differences in structural burden of disease. The findings strongly suggest biological equilibrium in SF composition between joint compartments in end-stage knee OA. Consequently, the anatomical site of SF collection is not an important practical implication for biomarker research in knee OA.