The two-stage revision protocol is the gold standard for controlling and treating low-grade prosthetic joint infections of total hip and total knee arthroplasty. The antibiotic pause for diagnostic reasons before reconstruction (stage two) is discussed in relation to the persistence of the infection and the development of resistant bacterial strains. Serological markers and a synovial analysis are commonly used to exclude the persistence of infection. Therefore, we asked (1) is the serological testing of C-reactive protein and leucocytes a valuable tool to predict a persistence of infection? and (2) what is the role of synovial aspiration of Plymethylmethacrylat (PMMA) spacers in hip and knee joints? One hundred twelve patients who were MSIS criteria-positive for a prosthetic joint infection were studied, including 45 total hip arthroplasties (THA) and 67 total knee artrhoplasties (TKA) patients. All patients were treated with a two-stage-protocol using a mobile PMMA spacer after a 14-day antibiotic-free interval, during which we measured serological markers (C-reactive protein and leucocytes) and performed synovial aspiration (white blood cell count, polymorphonuclear cell percentage, and microbiological culture) in these patients and compared the results with those of their long-term-follow-up (mean follow-up 27months, range 24-36months). Of the 112 patients, 89 patients (79.5%; 95% CI 72-86.9) exhibited infection control after a two-stage exchange, and we detected most methicillin-resistant, coagulase-negative Staphylococci (CoNS) in cases of a persistent infection. The mean sensitivity of serum C-reactive protein in the patients was 0.43 (range 0.23-0.64), and the mean specificity was 0.73 (range 0.64-0.82). For serum leucocytes, the mean sensitivity was 0.09 (range 0-0.29), and the mean specificity was 0.81 (range 0.7-0.92). The mean sensitivity for the WBC count in the synovial fluid (PMMA spacer aspiration) was 0.1 (range 0-0.29), and the mean specificity was 0.79 (range 0.68-0.92). For the PMN percentage, the mean sensitivity was 0.1 (range 0-0.29), and the mean specificity was 0.79 (range 0.68-0.92). No cut-off values could be established for C-reactive protein, leucocytes, WBC count and PMN percentage due to the low AUC. No reliable markers were identified for the long-term persistence of infection. C-reactive protein and leucocytes were often elevated, even when the infection was controlled. In addition, normalized serum markers did not exclude the persistence of infection during follow-up. The synovial analysis of the WBC count and PMN percentage did not predict the persistence of infection. However, microbiological synovial fluid analysis is often misleading due to false positive microbiological cultures, which results in overtreatment.