Background: Whole‐blood aggregometry (WBA) is a promising tool to assess platelet function in its physiological environment. Dilution of whole blood in WBA disregards platelet concentrations that may impact the results, especially in the case of low platelet counts. In a blinded, prospective in vitro study, the influence of platelet concentrations on WBA was assessed.Methods: Aggregation studies were carried out using whole blood from 10 healthy volunteers adjusted to platelet concentrations of 150, 100, 75, 50 and 25/nl using a plasma‐balanced crystalloid solution. Platelet aggregation was measured by a new near‐side whole blood aggregometer, activated by adenosin‐diphosphate, collagen and thrombin‐receptor activating protein. Three different approaches were applied: P1: whole blood diluted by an isotonic saline solution before activation, P2: undiluted whole blood with the single and P3: with the twofold concentration of the stimulating agent.Results: Aggregometry in diluted whole blood (P1) decreased significantly from a platelet concentration of 100/nl (P<0.01). In undiluted whole blood, aggregation declined significantly from concentrations of 75 and 50/nl for P2 and P3 (P<0.01). No correlation to platelet count occurred in the undiluted approaches until a platelet concentration of 75/nl, whereas correlation in the diluted test run was detected starting from 100/nl.Conclusions: This study demonstrates that WBA depends on the platelet count and sensitivity towards low platelet concentrations may be improved by abdication of further dilution and the use of undiluted whole blood.