D-dimer has been included in the criteria by the Musculoskeletal Infection Society in 2018 as a novel parameter to diagnose prosthetic joint infection (PJI). However, it is unclear how D-dimer levels change in between stages of a two-stage exchange. We prospectively investigated 30 patients who underwent a two-stage exchange using a spacer for PJI. D-Dimer, CRP and IL-6 were collected before first and second stage surgery and the difference (Δ) in between stages was calculated. The levels of plasma D-Dimer did not change from first to second stage surgery (2770 ng/ml (IQR, 1600–3770 ng/ml) versus 2340 ng/ml (IQR, 1270–4100 ng/ml); p = 0.8) while CRP (4.0 mg/dl (IQR, 1.7–5.5 mg/dl) versus 0.6 mg/dl (IQR, 0.5–0.8 mg/dl); p < 0.001) and IL-6 (21 pg/ml (IQR, 10–29 pg/ml) versus 6 pg/ml (4–9 pg/ml); p < 0.001) decreased. The ΔD-dimer between both stages was 300 ng/ml (range: − 2820 to 4280 ng/ml), the median ΔCRP was − 3.4 mg/dl (IQR, − 1.2 to − 4.8 mg/dl) and ΔIL-6 was − 13 pg/ml (IQR, − 4 to − 20 pg/ml). In 15 of 30 cases (50%) the D-dimer level increased between both stages, whereas the level of CRP (93%; 28/30) and IL-6 (96%; 28/29) decreased in most patients. As the level of serum D-dimers varies greatly, lacks a uniform decrease and does not identify persisting infection, surgeons should be cautious when using it at the timing of reimplantation.