Interleukin-1beta (IL-1beta) is a cytokine within the beta-trefoil family. Our data indicate that the folding/unfolding routes are geometrically frustrated. Follow-up theoretical studies predicted backtracking events that could contribute to the broad transition barrier and the experimentally observed long-lived intermediate. The backtracking route is attributed to the topological frustration introduced by the packing of the functional loop (the beta-bulge, residues 47-53) to the nascent barrel. We used real-time refolding NMR experiments to test for the presence of backtracking events predicted from our theoretical studies. Structural variants of IL-1beta, a beta-bulge deletion, and a circular permutation that opens the protein in the middle of the experimentally observed kinetic intermediate, were also refolded and studied to determine the affects on the observed folding reactions. The functional loop deletion variant demonstrated less backtracking than in WT protein whereas the permutation still maintains backtracking in agreement with theoretical predictions. Taken together, these findings indicate that the backtracking results from geometric frustration introduced into the fold for functional purposes.