Background Prolonged storage of transfused red blood cells (RBCs) is associated with hemolysis in healthy adults and inflammation in animal models. We aimed to determine whether storage duration affects markers of hemolysis (e.g., serum bilirubin, iron, and non-transferrin-bound iron (NTBI)) and inflammation (e.g., interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) in transfused very low birth weight (VLBW) infants. Methods Blood samples from 23 independent transfusion events were collected by heel stick before and 2–6h after transfusion. Results Serum iron, total bilirubin, NTBI, and MCP-1 levels were significantly increased after transfusion of RBCs (P<0.05 for each comparison). The storage age of transfused RBCs positively correlated with increases in NTBI following transfusion (P<0.001; R2 = 0.44). No associations between storage duration and changes in the other analytes were observed. Conclusions Transfusion of RBCs into VLBW infants is associated with increased markers of hemolysis and the inflammatory chemokine MCP-1. RBC storage duration only correlated with increases in NTBI levels following transfusion. NTBI was only observed in healthy adults following 35 days of storage; however, this study suggests that VLBW infants are potentially more susceptible to producing this pathological form of iron, with increased levels observed after transfusion of only 20-day old RBCs.