You have to create new item object instead of reusing it.
Try this :
protected void Page_Load(object sender, EventArgs e)
{
String User = Context.User.Identity.Name.Split(new Char[] { '\\' })[1];
MenuItem item = new MenuItem();
item.Text = "Configure";
item.NavigateUrl = "~/Default.aspx";
Menu1.Items.Add(item);
if (User.Equals(user1) | User.Equals(user2))
{
item = new MenuItem();
item.Text = "Appstore";
item.NavigateUrl = "~/AppStore.aspx";
Menu1.Items.Add(item);
}
item = new MenuItem();
item.Text = "About";
item.NavigateUrl = "~/About.aspx";
Menu1.Items.Add(item);
}