Before biological or biogeochemical experimentation, sediments are often manipulated and defaunated witb tbe use of many different approaches and only modest consideration of treatment effects on sediment biogeochemistry and fluxes. Mesocosm experiments require large quantities of sediment and no standard protocol to defaunate and equilibrate muddy sediments before initiation of experiments has been determined.Using fine-grained sediments, we examined a number of treatments: H) intact witb macroinfauna; (2) intact, macroinfauna individually removed; (3) homogenized surface sediment witb macrofauna; (4) bomogenized deep sediment witbout macroinfauna; and (5) intact deep sediment witbout macroinfauna. In weekly batcb core flux incubations, we measured dissolved oxygen, dinitrogen gas, ammonium (NH,;), nitrate plus nitrite (NO^ f NO,;), silicate (Si), and soluble reactive phospborus (SRP) fluxes over a 5-week period. In addition, we determined porewater ammcmium concentrations over time.Different sediment preparation techniques, witb the same muddy sediment, significantly affected nutrient and gas fluxes, and tbe amount of nutrient and gas fluxes differed between sediment preparation tecbniques. Severely manipulated sediments, sucb as homogenized treatments, had high initial effluxes but eventually equilibrated to lower and more constant nutrient and gas fluxes. Moreover, biogeocbemical flux cbanges for all treatments became similar after about 2 to 3 weeks. Sieved sediments exbibited low fluxes over tbe entire 5-week period, and flux rates did not cbange over time. A feasible method for sediment preparation for mesocosm studies is to use bomogenized deep sediment equilibrated over an almost 2-week period. Overall, sediment preparation and tbe time after sediment manipulation affect sediment biogeochemical processes and must be considered before initiating experiments.