The influence of tillage, carbon (C) and nitrogen (N) inputs and perennialization on labile fractions of soil organic matter (SOM) has been widely investigated, but research on the temporal and depth variation of labile C and N fractions in long‐term agroecosystems representative of the US Corn Belt is lacking. In this study, labile soil fractions as influenced by different agriculture management approaches common in the north‐central United States were characterized through measurements of potentially mineralizable N (PMN), mineralizable C (C‐min), and permanganate oxidizable C (POXC), which are known indicators of the biologically active pool of SOM. Soils were sampled five times in six different cropping systems at the Wisconsin Integrated Cropping Systems Trial: three annual cropping systems (continuous corn, strip‐till corn–soybean, organic grain) and three forage cropping systems (conventional forage, organic forage, grazed pasture). Across all sampling dates (May–September) and soil depth (0–15 and 15–30 cm), forage‐based cropping systems generally had greater PMN, C‐min, and POXC, with C‐min and PMN expressing the largest differences between forage and grain‐based cropping systems. Labile soil C and N fractions across cropping systems were typically greatest in July or August and were ∼50% greater in the top 0 to 15 cm of soil than the 15 to 30 cm. These results suggest sampling timing and depth for PMN, POXC, and C‐min need to be standardized to optimize their use as soil health indicators and that shifts in the system toward perenniality will be required to increase labile C and N fractions on the highly fertile Mollisols of the US Corn Belt.

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